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Sample records for microfluidic protein patterning

  1. Channel surface patterning of alternating biomimetic protein combinations for enhanced microfluidic tumor cell isolation.

    PubMed

    Launiere, Cari; Gaskill, Marissa; Czaplewski, Gregory; Myung, Ja Hye; Hong, Seungpyo; Eddington, David T

    2012-05-01

    Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

  2. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices.

    PubMed

    Lafleur, Josiane P; Kwapiszewski, Radoslaw; Jensen, Thomas G; Kutter, Jörg P

    2013-02-21

    The suitable optical properties of thiol-ene polymers combined with the ease of modifying their surface for the attachment of recognition molecules make them ideal candidates in many biochip applications. This paper reports the rapid one-step photochemical surface patterning of biomolecules in microfluidic thiol-ene chips. This work focuses on thiol-ene substrates featuring an excess of thiol groups at their surface. The thiol-ene stoichiometric composition can be varied to precisely control the number of surface thiol groups available for surface modification up to an average surface density of 136 ± 17 SH nm(-2). Biotin alkyne was patterned directly inside thiol-ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent wave-induced fluorescence (EWIF), demonstrating the success of the grafting procedure and its potential for biochip applications.

  3. Strategy for allosteric analysis based on protein-patterned stationary phase in microfluidic chip.

    PubMed

    Bi, Hongyan; Weng, Xuexiang; Qu, Haiyun; Kong, Jilie; Yang, Pengyuan; Liu, Baohong

    2005-01-01

    An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.

  4. Microfluidic Approaches for Protein Crystal Structure Analysis.

    PubMed

    Maeki, Masatoshi; Yamaguchi, Hiroshi; Tokeshi, Manabu; Miyazaki, Masaya

    2016-01-01

    This review summarizes two microfluidic-based protein crystallization methods, protein crystallization behavior in the microfluidic devices, and their applications for X-ray crystal structure analysis. Microfluidic devices provide many advantages for protein crystallography; they require small sample volumes, provide high-throughput screening, and allow control of the protein crystallization. A droplet-based protein crystallization method is a useful technique for high-throughput screening and the formation of a single crystal without any complicated device fabrication process. Well-based microfluidic platforms also enable effective protein crystallization. This review also summarizes the protein crystal growth behavior in microfluidic devices as, is known from viewpoints of theoretical and experimental approaches. Finally, we introduce applications of microfluidic devices for on-chip crystal structure analysis.

  5. Microfluidic Tools for Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Abdallah, Bahige G.

    X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm -- ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm -- ~20 ?m crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion

  6. Rapid Protein Separations in Microfluidic Devices

    NASA Technical Reports Server (NTRS)

    Fan, Z. H.; Das, Champak; Xia, Zheng; Stoyanov, Alexander V.; Fredrickson, Carl K.

    2004-01-01

    This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

  7. Integrated Microfluidics for Protein Modification Discovery.

    PubMed

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-10-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.

  8. Integrated Microfluidics for Protein Modification Discovery*

    PubMed Central

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-01-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research. PMID:26276765

  9. Microfluidic DNA extraction using a patterned aluminum oxide membrane

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Gale, Bruce K.

    2006-01-01

    A DNA extraction system was designed and fabricated using an AOM (aluminum oxide membrane) with 200 nm pores and PDMS microfluidic channels. The membrane was patterned using soft lithography techniques and SU-8 photolithography on the membrane. After making the pattern with SU-8, the AOM was observed using an SEM (scanning electro microscope) to verify the AOM structure was not damaged. From the SEM images, the AOM structure was not different after modification with SU-8. To complete the system, a PDMS mold for the microfluidic channels was made by soft lithography. Using the SU-8 mold, PDMS microchannels were cast using PDMS with a low polymer to curing agent ratio to provide adhesion between the patterned membrane and microfluidic channel. Then, the patterned membrane was sandwiched between PDMS microfluidic channels in a parallel format. The completed system was tested with 10ug of Lambda DNA mixed with the fluorescent dye SYBR Green I. Following DNA extraction, the surface of each well was examined with fluorescence microscopy while embedded in the microfluidic system. Extracted and immobilized DNA on the AOM was observed in almost every separation well. This microsystem, referred to as a membrane-on-a-chip, has potential applications in high-throughput DNA extraction and analysis, with the possibility of being integrated into polymer-based microfluidic systems.

  10. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  11. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  12. Structural characterization of soy protein nanoparticles from high shear microfluidization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein nanoparticles were produced with a microfluidizer and characterized in terms of particle size, size distribution, morphology, rheological properties, and aggregate structure. Three stages of structure breakdown were observed when the soy protein dispersion was passed through the microflu...

  13. Characterization of soy protein nanoparticles prepared by high shear microfluidization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein nanoparticles were produced with a microfluidizer and characterized in terms of particle size, size distribution, morphology, rheological properties, and aggregate structure. Three stages of structure breakdown were observed when the soy protein dispersion was passed through the microflu...

  14. Microfluidic Mixers for Studying Protein Folding

    PubMed Central

    Waldauer, Steven A.; Wu, Ling; Yao, Shuhuai; Bakajin, Olgica; Lapidus, Lisa J.

    2012-01-01

    The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4. Laminar flow is achieved for Reynolds numbers Re ≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM. The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms

  15. Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

    PubMed Central

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R2 = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays. PMID:23874811

  16. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  17. Protein Microarrays with Novel Microfluidic Methods: Current Advances.

    PubMed

    Dixit, Chandra K; Aguirre, Gerson R

    2014-07-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

  18. Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices.

    PubMed

    Kalish, Brent; Tsutsui, Hideaki

    2016-04-01

    We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

  19. Hydrogel microfluidics for the patterning of pluripotent stem cells

    PubMed Central

    Cosson, S.; Lutolf, M. P.

    2014-01-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate. PMID:24662945

  20. High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

    PubMed

    Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Ma, Chao; Zhao, Lei; Wang, Yaolei; Ouyang, Jia; Pang, Long; Wang, Jinyi

    2015-02-07

    Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PμSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PμS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PμSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PμS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PμS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PμS groups. We demonstrate that PμS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays.

  1. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host.

  2. Protein Microarrays with Novel Microfluidic Methods: Current Advances

    PubMed Central

    Dixit, Chandra K.; Aguirre, Gerson R.

    2014-01-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation. PMID:27600343

  3. Droplet-driven transports on superhydrophobic-patterned surface microfluidics.

    PubMed

    Xing, Siyuan; Harake, Ryan S; Pan, Tingrui

    2011-11-07

    Droplet-based transport phenomena driven by surface tension have been explored as an automated pumping source for a number of chemical and biological applications. In this paper, we present a comprehensive theoretical and experimental investigation of unconventional droplet-based motions on a superhydrophobic-patterned surface microfluidic (S(2)M) platform. The S(2)M surfaces are monolithically fabricated using a facile two-step laser micromachining technique on regular polydimethylsiloxane (PDMS) chemistry. Unlike the traditional droplet-driven pumps built on an enclosed microfluidic network, the S(2)M network pins the liquid-solid interface of droplets to the lithographically defined wetting boundary and establishes a direct linkage between the volumetric and hydraulic measures. Moreover, diverse modes of droplet motions are theoretically determined and experimentally characterized in a bi-droplet configuration, among which several unconventional droplet-driven transport phenomena are first demonstrated. These include big-to-small droplet merging, droplet balancing, as well as bidirectional transporting, in addition to the classic small-to-big droplet transition. Furthermore, multi-stage programmable bidirectional pumping has been implemented on the S(2)M platform, according to the newly established droplet manipulation principle, to illustrate its potential use for automated biomicrofluidic and point-of-care diagnostic applications.

  4. Protein Crystal Growth With the Aid of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark

    2003-01-01

    Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

  5. Patterning proteins and cells using soft lithography.

    PubMed

    Kane, R S; Takayama, S; Ostuni, E; Ingber, D E; Whitesides, G M

    1999-12-01

    This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

  6. Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

    PubMed

    Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

    2013-12-07

    In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

  7. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  8. Modular microfluidics for point-of-care protein purifications

    SciTech Connect

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

  9. Wettability control on multiphase flow in patterned microfluidics

    PubMed Central

    Zhao, Benzhong; Juanes, Ruben

    2016-01-01

    Multiphase flow in porous media is important in many natural and industrial processes, including geologic CO2 sequestration, enhanced oil recovery, and water infiltration into soil. Although it is well known that the wetting properties of porous media can vary drastically depending on the type of media and pore fluids, the effect of wettability on multiphase flow continues to challenge our microscopic and macroscopic descriptions. Here, we study the impact of wettability on viscously unfavorable fluid–fluid displacement in disordered media by means of high-resolution imaging in microfluidic flow cells patterned with vertical posts. By systematically varying the wettability of the flow cell over a wide range of contact angles, we find that increasing the substrate’s affinity to the invading fluid results in more efficient displacement of the defending fluid up to a critical wetting transition, beyond which the trend is reversed. We identify the pore-scale mechanisms—cooperative pore filling (increasing displacement efficiency) and corner flow (decreasing displacement efficiency)—responsible for this macroscale behavior, and show that they rely on the inherent 3D nature of interfacial flows, even in quasi-2D media. Our results demonstrate the powerful control of wettability on multiphase flow in porous media, and show that the markedly different invasion protocols that emerge—from pore filling to postbridging—are determined by physical mechanisms that are missing from current pore-scale and continuum-scale descriptions. PMID:27559089

  10. Wettability control on multiphase flow in patterned microfluidics

    NASA Astrophysics Data System (ADS)

    Juanes, Ruben; Zhao, Benzhong; MacMinn, Christopher

    2016-11-01

    Multiphase flow in porous media is important in many natural and industrial processes, including geologic CO2 sequestration, enhanced oil recovery, and water infiltration into soil. Although it is well known that the wetting properties of porous media can vary drastically depending on the type of media and pore fluids, the effect of wettability on multiphase flow continues to challenge our microscopic and macroscopic descriptions. Here, we study the impact of wettability on viscously unfavorable fluid-fluid displacement in disordered media by means of high-resolution imaging in microfluidic flow cells patterned with vertical posts. By systematically varying the wettability of the flow cell over a wide range of contact angles, we find that increasing the substrate's affinity to the injected fluid results in more efficient displacement of the defending fluid up to a critical wetting transition, beyond which the trend is reversed. We identify the pore-scale mechanisms-cooperative pore filling (increasing displacement efficiency) and corner flow (decreasing displacement efficiency)-responsible for this macroscale behavior, and show that they rely on the inherent 3D nature of interfacial flows, even in quasi-2D media. Our results demonstrate the powerful control of wettability on multiphase flow in porous media, and show that the markedly different invasion protocols that emerge-from pore-filling to post-bridging-are determined by physical mechanisms that are missing from current pore-scale and continuum-scale descriptions.

  11. Using Microfluidics to Decouple Nucleation and Growth of Protein Crystals.

    PubMed

    Shim, Jung-Uk; Cristobal, Galder; Link, Darren R; Thorsen, Todd; Fraden, Seth

    2007-01-01

    A high throughput, low volume microfluidic device has been designed to decouple the physical processes of protein crystal nucleation and growth. This device, called the Phase Chip, is constructed out of poly(dimethylsiloxane) (PDMS) elastomer. One of the Phase Chip's innovations is to exploit surface tension forces to guide each drop to a storage chamber. We demonstrate that nanoliter water-in-oil drops of protein solutions can be rapidly stored in individual wells thereby allowing the screening of 1000 conditions while consuming a total of only 10 mug protein on a 20 cm(2) chip. Another significant advance over current microfluidic devices is that each well is in contact with a reservoir via a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or protein. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current methods, such as the free interface diffusion or sitting drop methods, which are irreversible. The Phase Chip operates by first optimizing conditions for nucleation by using dialysis to supersaturate the protein solution, which leads to nucleation of many small crystals. Next, conditions are optimized for crystal growth by using dialysis to reduce the protein and precipitant concentrations, which leads small crystals to dissolve while simultaneously causing only the largest ones to grow, ultimately resulting in the transformation of many small, unusable crystals into a few large ones.

  12. One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

    PubMed Central

    Bavli, Danny; Ezra, Elishai; Kitsberg, Daniel; Murthy, Shashi K.; Nahmias, Yaakov

    2016-01-01

    Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation. PMID:27051469

  13. Pathogen receptor discovery with a microfluidic human membrane protein array.

    PubMed

    Glick, Yair; Ben-Ari, Ya'ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella; Gerber, Doron

    2016-04-19

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

  14. Model-controlled hydrodynamic focusing to generate multiple overlapping gradients of surface-immobilized proteins in microfluidic devices.

    PubMed

    Georgescu, Walter; Jourquin, Jerome; Estrada, Lourdes; Anderson, Alexander R A; Quaranta, Vito; Wikswo, John P

    2008-02-01

    Historically, it has been difficult to generate accurate and reproducible protein gradients for studies of interactions between cells and extracellular matrix. Here we demonstrate a method for rapid patterning of protein gradients using computer-driven hydrodynamic focusing in a simple microfluidic device. In contrast to published work, we are moving the complexity of gradient creation from the microfluidic hardware to dynamic computer control. Using our method, switching from one gradient profile to another requires only a few hours to devise a new control file, not days or weeks to design and build a new microfluidic device. Fitting existing protein deposition models to our data, we can extract key parameters needed for controlling protein deposition. Several protein deposition models were evaluated under microfluidic flow conditions. A mathematical model for our deposition method allows us to determine the parameters for a protein adsorption model and then predict the final shape of the surface density gradient. Simple and non-monotonic single and multi-protein gradient profiles were designed and deposited using the same device.

  15. Microfluidic Devices with Photodefinable Pseudo-valves for Protein Separation

    NASA Astrophysics Data System (ADS)

    Fan, Z. Hugh

    Plastic microfluidic devices are fabricated with an array of pseudo-valves for two-dimensional (2D) protein separation. The devices are made by compression molding; the mold is created by electroplating on a glass master fabricated by photolithography. Each device consists of one channel for isoelectric focusing (IEF) and multiple parallel channels for polyacrylamide gel electrophoresis (PAGE). The IEF channel (first dimension) is orthogonal to the PAGE channels (second dimension). Microfluidic pseudo-valves are created at the intersections of orthogonal channels by photodefinable, in situ gel polymerization. These valves enable the introduction of two types of separation media into orthogonal channels for performing 2D protein separation in the device. The presence of the pseudo-valves prevents one separation medium from being contaminated by the other medium, although proteins are allowed to transfer from the first to the second dimension under an electric field. Two-dimensional protein separation is achieved in less than 10 min, an improvement of two orders of magnitude compared with the conventional 2D gel electrophoresis using an IEF strip and a PAGE slab.

  16. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2005-02-10

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  17. Laminated microfluidic system for small sample protein analysis

    PubMed Central

    Saedinia, Sara; Nastiuk, Kent L.; Krolewski, John J.; Li, G. P.; Bachman, Mark

    2014-01-01

    We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons (“microfloupons”) that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation. PMID:24753728

  18. Effect of microfluidized and stearic acid modified soy protein in natural rubber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microfluidized and stearic acid modified soy protein aggregates were used to reinforced natural rubber. The size of soy protein particles was reduced with a microfluidizing and ball milling process. Filler size reduction with longer ball milling time tends to increase tensile strength of the rubber ...

  19. Modular microfluidics for point-of-care protein purifications

    DOE PAGES

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; ...

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

  20. Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine.

    PubMed

    Sechi, Deidre; Greer, Brady; Johnson, Jesse; Hashemi, Nastaran

    2013-11-19

    The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

  1. An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

    PubMed

    Choi, Jin-Woo; Oh, Kwang W; Thomas, Jennifer H; Heineman, William R; Halsall, H Brian; Nevin, Joseph H; Helmicki, Arthur J; Henderson, H Thurman; Ahn, Chong H

    2002-02-01

    This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

  2. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-20

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering.

  3. Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique.

    PubMed

    Nilghaz, Azadeh; Wicaksono, Dedy H B; Gustiono, Dwi; Abdul Majid, Fadzilah Adibah; Supriyanto, Eko; Abdul Kadir, Mohammed Rafiq

    2012-01-07

    This paper describes the fabrication of microfluidic cloth-based analytical devices (μCADs) using a simple wax patterning method on cotton cloth for performing colorimetric bioassays. Commercial cotton cloth fabric is proposed as a new inexpensive, lightweight, and flexible platform for fabricating two- (2D) and three-dimensional (3D) microfluidic systems. We demonstrated that the wicking property of the cotton microfluidic channel can be improved by scouring in soda ash (Na(2)CO(3)) solution which will remove the natural surface wax and expose the underlying texture of the cellulose fiber. After this treatment, we fabricated narrow hydrophilic channels with hydrophobic barriers made from patterned wax to define the 2D microfluidic devices. The designed pattern is carved on wax-impregnated paper, and subsequently transferred to attached cotton cloth by heat treatment. To further obtain 3D microfluidic devices having multiple layers of pattern, a single layer of wax patterned cloth can be folded along a predefined folding line and subsequently pressed using mechanical force. All the fabrication steps are simple and low cost since no special equipment is required. Diagnostic application of cloth-based devices is shown by the development of simple devices that wick and distribute microvolumes of simulated body fluids along the hydrophilic channels into reaction zones to react with analytical reagents. Colorimetric detection of bovine serum albumin (BSA) in artificial urine is carried out by direct visual observation of bromophenol blue (BPB) colour change in the reaction zones. Finally, we show the flexibility of the novel microfluidic platform by conducting a similar reaction in a bent pinned μCAD.

  4. Low-cost, high-throughput fabrication of cloth-based microfluidic devices using a photolithographical patterning technique.

    PubMed

    Wu, Peijing; Zhang, Chunsun

    2015-03-21

    In this work, we first report a facile, low-cost and high-throughput method for photolithographical fabrication of microfluidic cloth-based analytical devices (μCADs) by simply using a cotton cloth as a substrate material and employing an inexpensive hydrophobic photoresist laboratory-formulated from commercially available reagents, which allows patterning of reproducible hydrophilic-hydrophobic features in the cloth with well-defined and uniform boundaries. Firstly, we evaluated the wicking properties of cotton cloths by testing the wicking rate in the cloth channel, in combination with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses. It is demonstrated that the wicking properties of the cloth microfluidic channel can be improved by soaking the cloth substrate in 20 wt% NaOH solution and by washing the cloth-based microfluidic patterns with 3 wt% SDS solution. Next, we studied the minimum dimensions achievable for the width of the hydrophobic barriers and hydrophilic channels. The results indicate that the smallest width for a desired hydrophobic barrier is designed to be 100 μm and that for a desired hydrophilic channel is designed to be 500 μm. Finally, the high-throughput μCADs prepared using the developed fabrication technique were demonstrated for colorimetric assays of glucose and protein in artificial urine samples. It has been shown that the photolithographically patterned μCADs have potential for a simple, quantitative colorimetric urine test. The combination of cheap cloth and inexpensive high-throughput photolithography enables the development of new types of low-cost cloth-based microfluidic devices, such as "microzone plates" and "gate arrays", which provide new methods to perform biochemical assays or control fluid flow.

  5. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    NASA Astrophysics Data System (ADS)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  6. Direct optical patterning of poly(dimethylsiloxane) microstructures for microfluidic chips

    NASA Astrophysics Data System (ADS)

    Gao, Shaorui; Tung, Wing-Tai; Wong, Dexter S.; Bian, Liming; Zhang, A. Ping

    2016-10-01

    In this paper, we present an optical maskless exposure approach for direct patterning of large-area high resolution microfluidic chips using photosensitive poly(dimethylsiloxane) (PDMS) materials. Both positive- and negative-tone photosensitive PDMS (photoPDMS) were successfully patterned into various microfluidic devices with complex geometries by using an optical maskless lithography process. The positive-tone PDMS is used for patterning of largearea chips, while the negative-tone PDMS is demonstrated to fabricate high-resolution microstructures and on-chip devices. With the seamless pattern-stitching technique, a large-area microfluidic chip with size of 5.5 × 2.8 cm2 with complex three-dimensional (3D) staggered herringbone mixers (SHMs) for micro-flow gradient generation has been directly fabricated within 125 minutes by using the positive-tone PDMS. A small microfluidic chip with feature size as small as 5 μm is demonstrated by using the negative-tone PDMS. The experimental results reveal that the optical maskless lithography technology enables to rapidly pattern high-resolution microstructures and is very promising for development of lab-on-a-chip devices.

  7. Microfluidic Western Blotting of Low-Molecular-Mass Proteins

    PubMed Central

    2015-01-01

    We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. PMID:25268977

  8. Pathogen receptor discovery with a microfluidic human membrane protein array

    PubMed Central

    Glick, Yair; Ben-Ari, Ya’ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella

    2016-01-01

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein–pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism. PMID:27044079

  9. Regioselective patterning of multiple SAMs and applications in surface-guided smart microfluidics.

    PubMed

    Chen, Chuanzhao; Xu, Pengcheng; Li, Xinxin

    2014-12-24

    A top-down nanofabrication technology is developed to integrate multiple SAMs (self-assembled monolayers) into regioselective patterns. With ultraviolet light exposure through regioselectively hollowed hard mask, an existing SAM at designated microregions can be removed and a dissimilar kind of SAM can be regrown there. By repeating the photolithography-like process cycle, diverse kinds of SAM building blocks can be laid out as a desired pattern in one microfluidic channel. In order to ensure high quality of the surface modifications, the SAMs are vapor-phase deposited before the channel is closed by a bonding process. For the first time the technique makes it possible to integrate three or more kinds of SAMs in one microchannel. The technique is very useful for multiplex surface functionalization of microfluidic chips where different segments of a microfluidic channel need to be individually modified with different SAMs or into arrayed pattern for surface-guided fluidic properties like hydrophobicity/philicity and/or oleophobicity/philicity, etc. The technique has been well validated by experimental demonstration of various surface-directed flow-guiding functions. By modifying a microchannel surface into an arrayed pattern of multi-SAM "two-tone" stripe array, surface-guiding-induced 3D swirling flow is generated in a microfluidic channel that experimentally exhibits quick oil/water mixing and high-efficiency oil-to-water chemical extraction.

  10. Microfluidic Patterning of Metal Structures for Flexible Conductors by In Situ Polymer-Assisted Electroless Deposition.

    PubMed

    Liang, Suqing; Li, Yaoyao; Zhou, Tingjiao; Yang, Jinbin; Zhou, Xiaohu; Zhu, Taipeng; Huang, Junqiao; Zhu, Julie; Zhu, Deyong; Liu, Yizhen; He, Chuanxin; Zhang, Junmin; Zhou, Xuechang

    2017-02-01

    A low-cost, solution-processed, versatile, microfluidic approach is developed for patterning structures of highly conductive metals (e.g., copper, silver, and nickel) on chemically modified flexible polyethylene terephthalate thin films by in situ polymer-assisted electroless metal deposition. This method has significantly lowered the consumption of catalyst as well as the metal plating solution.

  11. Identification of microfluidic two-phase flow patterns in lab-on-chip devices.

    PubMed

    Yang, Zhaochu; Dong, Tao; Halvorsen, Einar

    2014-01-01

    This work describes a capacitive sensor for identification of microfluidic two-phase flow in lab-on-chip devices. With interdigital electrodes and thin insulation layer utilized, this sensor is capable of being integrated with the microsystems easily. Transducing principle and design considerations are presented with respect to the microfluidic gas/liquid flow patterns. Numerical simulation results verify the operational principle. And the factors affecting the performance of the sensor are discussed. Besides, a feasible process flow for the fabrication is also proposed.

  12. Fabrication of microfluidic devices containing patterned microwell arrays.

    PubMed

    Henley, W Hampton; Dennis, Patty J; Ramsey, J Michael

    2012-02-07

    A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 μm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 μm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 μm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 μm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.

  13. "Print-n-Shrink" technology for the rapid production of microfluidic chips and protein microarrays.

    PubMed

    Sollier, Kevin; Mandon, Céline A; Heyries, Kevin A; Blum, Loïc J; Marquette, Christophe A

    2009-12-21

    An innovative method for the production of microfluidic chips integrating protein spots is described. The technology, called "Print-n-Shrink", is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink polystyrene sheets. The initial print which has a minimum size of 15 microm (height) x 230 microm (width) is thermally treated (30 seconds, 163 degrees C) to shrink and generate features of 85 microm (height) x 100 microm (width). Concomitantly, proteins such as monoclonal antibodies or cellular adhesion proteins are spotted onto the Polyshrink sheets and shrunk together with the microfluidic design, creating a complete biochip integrating both complex microfluidic designs and protein spots for bioanalytical applications.

  14. A novel microfluidics-based method for probing weak protein-protein interactions.

    PubMed

    Tan, Darren Cherng-wen; Wijaya, I Putu Mahendra; Andreasson-Ochsner, Mirjam; Vasina, Elena Nikolaevna; Nallani, Madhavan; Hunziker, Walter; Sinner, Eva-Kathrin

    2012-08-07

    We report the use of a novel microfluidics-based method to detect weak protein-protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein-protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein-protein interactions, particularly between membrane proteins.

  15. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    NASA Astrophysics Data System (ADS)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  16. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  17. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-07

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity.

  18. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion

    NASA Astrophysics Data System (ADS)

    Hansen, Carl L.; Skordalakes, Emmanuel; Berger, James M.; Quake, Stephen R.

    2002-12-01

    Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

  19. Integration of protein processing steps on a droplet microfluidics platform for MALDI-MS analysis.

    PubMed

    Chatterjee, Debalina; Ytterberg, A Jimmy; Son, Sang Uk; Loo, Joseph A; Garrell, Robin L

    2010-03-01

    A droplet-based (digital) microfluidics platform has been developed to prepare and purify protein samples for measurement by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Liquid droplets are moved in air by sequentially applying an electric potential to an array of electrodes patterned beneath a hydrophobic dielectric layer. We show that a complete integrated sequence of protein processing steps can be performed on this platform, including disulfide reduction, alkylation, and enzymatic digestion, followed by cocrystallization with a MALDI matrix and analysis of the sample in situ by MALDI-MS. Proteins carbonic anhydrase, cytochrome c, and ubiquitin were used to demonstrate the digestion and postdigestion steps; insulin, serum albumin, and lysozyme were used to illustrate the complete sequence of protein processing steps available with the platform. Several functional improvements in the platform are reported, notably, the incorporation of acetonitrile in the protein droplets to facilitate movement, and patterning the device surfaces to optimize sample crystallization. The method is fast, simple, repeatable, and results in lower reagent consumption and sample loss than conventional techniques for proteomics sample preparation.

  20. Tape Transfer Atomization Patterning of Liquid Alloys for Microfluidic Stretchable Wireless Power Transfer

    PubMed Central

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-01-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times. PMID:25673261

  1. Protein crystallization using microfluidic technologies based on valves, droplets, and SlipChip.

    PubMed

    Li, Liang; Ismagilov, Rustem F

    2010-01-01

    To obtain protein crystals, researchers must search for conditions in multidimensional chemical space. Empirically, thousands of crystallization experiments are carried out to screen various precipitants at multiple concentrations. Microfluidics can manipulate fluids on a nanoliter scale, and it affects crystallization twofold. First, it miniaturizes the experiments that can currently be done on a larger scale and enables crystallization of proteins that are available only in small amounts. Second, it offers unique experimental approaches that are difficult or impossible to implement on a larger scale. Ongoing development of microfluidic techniques and their integration with protein production, characterization, and in situ diffraction promises to accelerate the progress of structural biology.

  2. Fabrication of thermo-responsive microfluidic membrane using photopolymerization patterning

    NASA Astrophysics Data System (ADS)

    Kim, Hyejeong; Lee, Sang Joon

    2015-11-01

    The programmed manipulation of responsive functional hydrogels is receiving large attention because of its unique functions and wide range of engineering applications. In this study, we developed an innovative stomata-inspired membrane (SIM) by fabricating a temperature-responsive hydrogel with a simple, cost effective, and high-throughput photopolymerization patterning process. Polymerization-induced diffusion on the macro-scale surface gives rise to form a multi-parted polymer membrane with fine pores by simple UV irradiation. After heating the SIM, the less deformable thick frame supports the whole structure, and the highly deformable thin base regulates the size of pores. The morphological configuration of the SIM can be easily changed by varying the solution composition or selecting a suitable photomask with different pattern. The developed SIM has the special sensing-to-actuation functions of stimuli-responsive hydrogels. This membrane with temperature-responsive pores would be potentially utilized in numerous practical applications, such as filter membranes with self-adjustable pores, membrane-based sensors, membrane-based actuators, and multi-functional membranes etc. This study was supported by the National Research Foundation of Korea (NRF) and funded by the Korean government (MSIP) (Grant No. 2008-0061991).

  3. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-04-01

    A microfluidic platform has been developed for the capture and X-ray analysis of protein microcrystals, affording a means to improve the efficiency of XFEL and synchrotron experiments. X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  4. Understanding wax screen-printing: a novel patterning process for microfluidic cloth-based analytical devices.

    PubMed

    Liu, Min; Zhang, Chunsun; Liu, Feifei

    2015-09-03

    In this work, we first introduce the fabrication of microfluidic cloth-based analytical devices (μCADs) using a wax screen-printing approach that is suitable for simple, inexpensive, rapid, low-energy-consumption and high-throughput preparation of cloth-based analytical devices. We have carried out a detailed study on the wax screen-printing of μCADs and have obtained some interesting results. Firstly, an analytical model is established for the spreading of molten wax in cloth. Secondly, a new wax screen-printing process has been proposed for fabricating μCADs, where the melting of wax into the cloth is much faster (∼5 s) and the heating temperature is much lower (75 °C). Thirdly, the experimental results show that the patterning effects of the proposed wax screen-printing method depend to a certain extent on types of screens, wax melting temperatures and melting time. Under optimized conditions, the minimum printing width of hydrophobic wax barrier and hydrophilic channel is 100 μm and 1.9 mm, respectively. Importantly, the developed analytical model is also well validated by these experiments. Fourthly, the μCADs fabricated by the presented wax screen-printing method are used to perform a proof-of-concept assay of glucose or protein in artificial urine with rapid high-throughput detection taking place on a 48-chamber cloth-based device and being performed by a visual readout. Overall, the developed cloth-based wax screen-printing and arrayed μCADs should provide a new research direction in the development of advanced sensor arrays for detection of a series of analytes relevant to many diverse applications.

  5. Acoustic Tweezing and Patterning of Concentration Fields in Microfluidics

    NASA Astrophysics Data System (ADS)

    Karlsen, Jonas T.; Bruus, Henrik

    2017-03-01

    We demonstrate theoretically that acoustic forces acting on inhomogeneous fluids can be used to pattern and manipulate solute concentration fields into spatiotemporally controllable configurations stabilized against gravity. A theoretical framework describing the dynamics of concentration fields that weakly perturb the fluid density and speed of sound is presented and applied to study manipulation of concentration fields in rectangular-channel acoustic eigenmodes and in Bessel-function acoustic vortices. In the first example, methods to obtain horizontal and vertical multilayer stratification of the concentration field at the end of a flow-through channel are presented. In the second example, we demonstrate acoustic tweezing and spatiotemporal manipulation of a local high-concentration region in a lower-concentration medium, thereby extending the realm of acoustic tweezing to include concentration fields.

  6. Refolding of difficult-to-fold proteins by a gradual decrease of denaturant using microfluidic chips.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya; Briones-Nagata, Maria Portia; Maeda, Hideaki

    2010-06-01

    Protein refolding is an important process to obtain active recombinant proteins from inclusion bodies (protein aggregates). However, the conventional refolding method of dialysis or dilution is a time consuming procedure and often, recovering yields of active proteins are low. In this study, we used controllable diffusion through laminar flow in microchannels to control the denaturant concentration. The performance of the designed microfluidic chips was evaluated by the refolding of difficult-to-fold proteins (citrate synthase and the zeta-associated protein 70-kDa protein kinase domain). We demonstrated this by varying the flow rates of the diluting buffer stream(s) and multi-junctions which could control the different flow rate ratios of the buffer stream(s) and the denatured protein stream. By this strategy, we were able to improve the efficiency of protein refolding. Our method achieved refolding within a short period of time at room temperature without the need of any small molecules or chaperone proteins. Moreover, the efficiency of protein refolding by microfluidic chip was found higher than that prepared by dialysis or dilution. These results suggest that microfluidic chips employing this strategy may provide miniaturized tools for rapid and efficient recovery of active proteins from inclusion bodies.

  7. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    PubMed Central

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  8. TiO2 coated microfluidic devices for recoverable hydrophilic and hydrophobic patterns

    NASA Astrophysics Data System (ADS)

    Lee, Jin-Hyung; Kim, Sang Kyung; Park, Hyung-Ho; Kim, Tae Song

    2015-03-01

    We report a simple method for modifying the surfaces of plastic microfluidic devices through dynamic coating process with a nano-colloidal TiO2 sol. The surface of the thermoplastic, cyclic olefin copolymer (COC) was coated with the TiO2 film, that displayed an effective photocatalytic property. The hydrophilic surface is obtained in the TiO2-coated zone of a microfluidic channel, and TiO2 coated surface degradation can be reversed easily by UV irradiation. The present work shows a photocatalytic activity concerning the effect of TiO2 coating density, which is controlled by the number of coating cycles. The hydrophilized surface was characterized by the contact angle of water and the TiO2 coated COC surface reduced the water contact angle from 85° to less than 10° upon UV irradiation. The photocatalytic effect of the layer that was coated five times with TiO2 was excellent, and the super-hydrophilicity of the TiO2 surface could be promptly recovered after 10 months of storage at atmospheric conditions. The COC microfluidic devices, in which TiO2 has been freshly deposited and aged for 10 months, were capable of generating water-in oil-in water (W/O/W) double emulsions easily and uniformly by simple control of the flow rates for demonstration of excellent hydrophilic patterning and recovery of the TiO2 coated in the microchannels.

  9. Microfluidics for the analysis of membrane proteins: how do we get there?

    PubMed

    Battle, Katrina N; Uba, Franklin I; Soper, Steven A

    2014-08-01

    The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented.

  10. Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

    PubMed

    Sang, Chen-Hsiang; Chou, Shu-Jen; Pan, F M; Sheu, Jeng-Tzong

    2016-01-15

    In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

  11. Flexible method for fabricating protein patterns on superhydrophobic platforms controlled by magnetic field.

    PubMed

    Wang, Jian; Li, Hao; Zou, Haoyang; Wang, Chenmiao; Zhang, Hao; Mano, João F; Song, Wenlong

    2017-02-28

    Inspired by the rolling of water droplets on lotus leaves, we developed a novel, magnetic field-controlled patterning method for water-soluble proteins and other functional materials on superhydrophobic platforms. This simple method can be used to fabricate biochips and open micro-fluidic devices in a simple way.

  12. Patterned solvent delivery and etching for the fabrication of plastic microfluidic devices.

    PubMed

    Brister, Paul C; Weston, Kenneth D

    2005-11-15

    A very simple method for micropatterning flat plastic substrates that can be used to build microfluidic devices is demonstrated. Patterned poly(dimethylsiloxane) elastomer is used as a template to control the flow path of an etching solvent through a channel design to be reproduced on the plastic substrate. The etching solvent was a acetone/ethanol mixture for poly(methyl methacrylate) substrates or a dimethylformamide/acetone mixture for polystyrene. The method is extremely fast in that duplicate plastic substrates can be patterned in just a few minutes each. We identified conditions that lead to smooth channel surfaces and characterized the rate of etching under these conditions. We determined that, for sufficiently short etching times (shallow channel depths), the etch rate is independent of the linear flow rate. This is very important since it means that the etch depth is approximately constant even in complex channel geometries where there will be a wide range of etchant flow rates at different positions in the pattern to be reproduced. We also demonstrate that the method can be used to produce channels with different depths on the same substrate as well as channels that intersect to form a continuous fluid junction. The method provides a nice alternative to existing methods to rapidly fabricate microfluidic devices in rigid plastics without the need for specialized equipment.

  13. Application of microfluidic chip with integrated optics for electrophoretic separations of proteins.

    PubMed

    Vieillard, Julien; Mazurczyk, Radoslaw; Morin, Christophe; Hannes, Benjamin; Chevolot, Yann; Desbène, Paul-Louis; Krawczyk, Stanislas

    2007-01-15

    This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.

  14. A Novel Impedimetric Microfluidic Analysis System for Transgenic Protein Cry1Ab Detection

    PubMed Central

    Jin, Shunru; Ye, Zunzhong; Wang, Yixian; Ying, Yibin

    2017-01-01

    Impedimetric analysis method is an important tool for food safety detection. In this work, a novel impedimetric microfluidic analysis system consisted of a printed gold electrode chip and a microfluidic flow cell was developed for sensitive and selective detection of transgenic protein Cry1Ab. Anti-Cry1Ab aptamer coated magnetic beads were used to recognize transgenic protein Cry1Ab and form Cry1Ab-aptamer modified magnetic beads. After separation, the obtained Cry1Ab-aptamer modified magnetic beads were dissolved in 0.01 M mannitol and followed by injection into the microfluidic flow cell for impedimetric measurement. At the frequency of 358.3 Hz, the impedance signal shows a good linearity with the concentrations of Cry1Ab protein at a range from 0 to 0.2 nM, and the detection limit is 0.015 nM. The results demonstrate that the impedimetric microfluidic analysis system provides an alternative way to enable sensitive, rapid and specific detection of transgenic protein Cry1Ab. PMID:28251986

  15. A Microfluidic Device for Immunoassay-Based Protein Analysis of Single E. coli Bacteria.

    PubMed

    Stratz, Simone; Dittrich, Petra S

    2015-01-01

    We present a method suitable for quantitative analysis of intracellular proteins, metabolites and secondary messengers of single bacterial cells. The method integrates the concept of immunoassays on a microfluidic device that facilitates single cell trapping and isolating in a small volume of a few tens of picoliters. Combination of the benefits of microfluidic systems for single cell analysis with the high analytical selectivity and sensitivity of immunoassays enables the detection of even low abundant intracellular analytes which occur only at a few hundred copies per bacterium.

  16. Optically Induced Thermal Gradients for Protein Characterization in Nanolitre-scale Samples in Microfluidic Devices

    PubMed Central

    Sagar, D. M.; Aoudjane, Samir; Gaudet, Matthieu; Aeppli, Gabriel; Dalby, Paul A.

    2013-01-01

    Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry. PMID:23823279

  17. Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics.

    PubMed

    Lucchetta, Elena M; Lee, Ji Hwan; Fu, Lydia A; Patel, Nipam H; Ismagilov, Rustem F

    2005-04-28

    Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions--a temperature step--in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100 min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development.

  18. Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics

    NASA Astrophysics Data System (ADS)

    Lucchetta, Elena M.; Lee, Ji Hwan; Fu, Lydia A.; Patel, Nipam H.; Ismagilov, Rustem F.

    2005-04-01

    Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions-a temperature step-in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development.

  19. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction.

    PubMed

    Emamzadah, Soheila; Petty, Tom J; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean Luc; Halazonetis, Thanos D

    2009-09-01

    Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 A resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

  20. Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography.

    PubMed

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei; Yuan, Hua

    2016-07-01

    Microfluidic chip electrophoresis (MCE) is a powerful separation tool for biomacromolecule analysis. However, adsorption of biomacromolecules, particularly proteins onto microfluidic channels severely degrades the separation performance of MCE. In this paper, an anti-protein-fouling MCE was fabricated using a novel sandwich photolithography of poly(ethylene glycol) (PEG) prepolymers. Photopatterned microchannel with a minimum resolution of 10 μm was achieved. After equipped with a conventional online electrochemical detector, the device enabled baseline separation of bovine serum albumin, lysozyme (Lys), and cytochrome c (Cyt-c) in 53 s under a voltage of 200 V. Compared with a traditional polydimethylsiloxane MCE made by soft lithography, the PEG MCE made by the sandwich photolithography not only eliminated the need of a master mold and the additional modification process of the microchannel but also showed excellent anti-protein-fouling properties for protein separation.

  1. Fabrication of microfluidic chips using lithographic patterning and adhesive bonding of the thick negative photoresist AZ 125 nXT

    NASA Astrophysics Data System (ADS)

    Knoll, Thorsten; Bergmann, Andreas; Nußbaum, Dominic

    2015-05-01

    In this work, for the first time the negative photoresist AZ 125 nXT was used for the fabrication of a microfluidic chip. Usually, fabrication of microfluidic devices on the basis of silicon or glass substrates is done by using the epoxy-based negative photoresist SU-8 or other thick film polymer materials. The suitability of SU-8 for various microfluidic applications has been shown in the fields of bioanalytic devices, lab-on-chip systems or microreaction technology. However, processing is always a very challenging task with regard to the adaptation of process parameters to the individual design and required functionality. Now, the AZ 125 nXT allows for the fabrication of structures in a wide thickness range with only one type of viscosity. In contrast to SU-8, the AZ 125 nXT is fully cross-linked during UV exposure and does not require a time-consuming post-exposure bake. 90 μm deep microfluidic channels were defined by lithographic patterning of AZ 125 nXT. Sealing of the open microfluidic channels was performed by a manual adhesive bonding process at a temperature of 100 °C. The fluidic function was successfully tested with flow rates up to 20 ml/min by means of a microfluidic edge connector. Long term stability and chemical resistance of the fabricated microfluidic channels will be investigated in the near future. The presented work shows the potential of AZ 125 nXT as a possible alternative to SU-8 for the fabrication of microfluidic chips.

  2. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

    PubMed Central

    Mozdzierz, Nicholas J.; Love, Kerry R.; Lee, Kevin S.; Lee, Harry L. T.; Shah, Kartik A.; Ram, Rajeev J.

    2015-01-01

    We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing. PMID:26055071

  3. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    SciTech Connect

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-09-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

  4. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array.

    PubMed

    Lyubimov, Artem Y; Murray, Thomas D; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Cohen, Aina E; Soltis, S Michael; Baxter, Elizabeth L; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Berger, James M

    2015-04-01

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  5. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  6. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    DOE PAGES

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; ...

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat formore » conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.« less

  7. Rapid cell-patterning and microfluidic chip fabrication by crack-free CO2 laser ablation on glass

    NASA Astrophysics Data System (ADS)

    Yen, Meng-Hua; Cheng, Ji-Yen; Wei, Cheng-Wey; Chuang, Yung-Chuan; Young, Tai-Horng

    2006-07-01

    This paper uses a widely available CO2 laser scriber (λ = 10.6 µm) to perform the direct-writing ablation of quartz, borofloat and pyrex substrates for the development of microfluidic chips and cell chips. The surface quality of the ablated microchannels and the presence of debris and distortion are examined by scanning electron microscopy, atomic force microscopy and surface profile measurement techniques. The developed laser ablation system provides a versatile and economic approach for the fabrication of glass microfluidic chips with crack-free structures. In the laser writing process, the desired microfluidic patterns are designed using commercial computer software and are then transferred to the laser scriber to ablate the trenches. This process eliminates the requirement for corrosive chemicals and photomasks, and hence the overall microchip development time is limited to less than 24 h. Additionally, since the laser writing process is not limited by the dimensions of a photomask, the microchannels can be written over a large substrate area. The machining capability and versatility of the laser writing system are demonstrated through its application to the fabrication of a borofloat microfluidic chip and the writing of a series of asymmetric trenches in a microwell array. It is shown that the minimum attainable trench width is 95 µm and that the maximum trench depth is 225 µm. The system provides an economic and powerful means of rapid glass microfluidic chip development. A rapid cell-patterning method based on this method is also demonstrated.

  8. A microfluidic device for multiplexed protein detection in nano-liter volumes.

    PubMed

    Diercks, Alan H; Ozinsky, Adrian; Hansen, Carl L; Spotts, James M; Rodriguez, David J; Aderem, Alan

    2009-03-01

    We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nano-liter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100-microm(2) sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nano-liter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of tumor necrosis factor (TNF) in a volume of 4.7nl.

  9. Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy.

    PubMed

    Kane, Avinash S; Hoffmann, Armin; Baumgärtel, Peter; Seckler, Robert; Reichardt, Gerd; Horsley, David A; Schuler, Benjamin; Bakajin, Olgica

    2008-12-15

    We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 micros. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.

  10. Specific transport of target molecules by motor proteins in microfluidic channels.

    PubMed

    Tarhan, Mehmet C; Yokokawa, Ryuji; Morin, Fabrice O; Fujita, Hiroyuki

    2013-06-03

    Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor-protein-driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom-up functionality of biological systems with the top-down handling capabilities of micro-electromechanical systems technology, highly selective molecular recognition and motor-protein-based transport is integrated in a microfluidic channel network.

  11. Proteolysis in microfluidic droplets: an approach to interface protein separation and peptide mass spectrometry.

    PubMed

    Ji, Ji; Nie, Lei; Qiao, Liang; Li, Yixin; Guo, Liping; Liu, Baohong; Yang, Pengyuan; Girault, Hubert H

    2012-08-07

    A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.

  12. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  13. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  14. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.

    PubMed

    Pessi, Jenni; Santos, Hélder A; Miroshnyk, Inna; JoukoYliruusi; Weitz, David A; Mirza, Sabiruddin

    2014-09-10

    In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23-47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of BSA in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

  15. A Versatile Method of Patterning Proteins and Cells.

    PubMed

    Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

    2017-02-26

    Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

  16. A microfluidic approach for protein structure determination at room temperature via on-chip anomalous diffraction.

    PubMed

    Perry, Sarah L; Guha, Sudipto; Pawate, Ashtamurthy S; Bhaskarla, Amrit; Agarwal, Vinayak; Nair, Satish K; Kenis, Paul J A

    2013-08-21

    We report a microfluidic approach for de novo protein structure determination via crystallization screening and optimization, as well as on-chip X-ray diffraction data collection. The structure of phosphonoacetate hydrolase (PhnA) has been solved to 2.11 Åvia on-chip collection of anomalous data that has an order of magnitude lower mosaicity than what is typical for traditional structure determination methods.

  17. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.

    PubMed

    Davis, Lloyd M; Lubbeck, Jennifer L; Dean, Kevin M; Palmer, Amy E; Jimenez, Ralph

    2013-06-21

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.

  18. Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting.

    PubMed

    Kim, Dohyun; Karns, Kelly; Tia, Samuel Q; He, Mei; Herr, Amy E

    2012-03-06

    We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren's Syndrome, an autoimmune disease.

  19. Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

    PubMed Central

    Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2014-01-01

    Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

  20. Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.

    PubMed

    Zhu, Ying; Zhu, Li-Na; Guo, Rui; Cui, Heng-Jun; Ye, Sheng; Fang, Qun

    2014-05-23

    Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35-96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4-8 nL. The protein consumption significantly reduces 50-500 fold compared with current crystallization stations.

  1. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    PubMed Central

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-01-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

  2. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    NASA Astrophysics Data System (ADS)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  3. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

    PubMed

    Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

    2017-03-14

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  4. An integrated microfluidics-tandem mass spectrometry system for automated protein analysis.

    PubMed

    Figeys, D; Gygi, S P; McKinnon, G; Aebersold, R

    1998-09-15

    We describe an integrated analytical system consisting of a microfluidics device micromachined using photolithography/etching technology, a panel of computer-controlled high-voltage relays, and an electrospray ionization tandem mass spectrometer. Movement of solvents and samples on the device and off the device to the mass spectrometer was achieved by directed electroosmotic pumping induced by the activation of a suitable constellation of high-voltage relays. The system was used for the sequential automated analysis of protein digests. We demonstrate low femtomole per microliter sensitivity of detection and compatibility of the system with the automated analysis of proteins separated by two-dimensional gel electrophoresis.

  5. Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

    NASA Astrophysics Data System (ADS)

    Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

    2012-02-01

    Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

  6. Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

    PubMed Central

    Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison. E.; Han, Jongyoon; Alter, Galit

    2016-01-01

    Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets–cells or proteins–bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients. PMID:27026280

  7. Microfluidic polyacrylamide gel electrophoresis with in situ immunoblotting for native protein analysis.

    PubMed

    He, Mei; Herr, Amy E

    2009-10-01

    We introduce an automated immunoblotting method that reports protein electrophoretic mobility and identity in a single streamlined microfluidic assay. Native polyacrylamide gel electrophoresis (PAGE) was integrated with subsequent in situ immunoblotting. Integration of three PA gel elements into a glass microfluidic chip achieved multiple functions, including (1) rapid protein separation via on-chip PAGE, (2) directed electrophoretic transfer of resolved protein peaks to an in-line blotting membrane, and (3) high-efficiency identification of the transferred proteins using antibody-functionalized blotting membranes. In-chip blotting membranes were photopatterned with biotinylated antibody using streptavidin polyacrylamide (PA) thus yielding postseparation sample analysis. No pressure driven flow or fluid valving was required, as the assay was operated by electrokinetically programmed control. A model sample of fluorescently labeled BSA (negative control), alpha-actinin, and prostate specific antigen (PSA) was selected to develop and characterize the assay. A 5 min assay time was required without operator intervention. Optimization of the blotting membrane (geometry, operation, and composition) yielded a detection limit of approximately 0.05 pg (alpha-actinin peak). An important additional blotting fabrication strategy was developed and characterized to allow vanishingly small antibody consumption (approximately 1 microg), as well as end-user customization of the blotting membrane after device fabrication and storage. This first report of rapid on-chip protein PAGE integrated with in situ immunoblotting forms the basis for a sensitive, automated approach applicable to numerous forms of immunoblotting.

  8. A Microfluidic Platform for Characterization of Protein—Protein Interactions

    PubMed Central

    Javanmard, Mehdi; Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Huber, David E.; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W.

    2010-01-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein–protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein–glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  9. Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization

    PubMed Central

    Zheng, Bo; Gerdts, Cory J; Ismagilov, Rustem F

    2006-01-01

    Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs —aqueous droplets surrounded by a fluorinated carrier fluid —have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals. PMID:16154351

  10. A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening.

    PubMed

    Li, Gang; Chen, Qiang; Li, Junjun; Hu, Xiaojian; Zhao, Jianlong

    2010-06-01

    A centrifuge-based microfluidic system has been developed that enables automated high-throughput and low-volume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control.

  11. Paper-based microfluidic approach for surface-enhanced raman spectroscopy and highly reproducible detection of proteins beyond picomolar concentration.

    PubMed

    Saha, Arindam; Jana, Nikhil R

    2015-01-14

    Although microfluidic approach is widely used in various point of care diagnostics, its implementation in surface enhanced Raman spectroscopy (SERS)-based detection is challenging. This is because SERS signal depends on plasmonic nanoparticle aggregation induced generation of stable electromagnetic hot spots and in currently available microfluidic platform this condition is difficult to adapt. Here we show that SERS can be adapted using simple paper based microfluidic system where both the plasmonic nanomaterials and analyte are used in mobile phase. This approach allows analyte induced controlled particle aggregation and electromagnetic hot spot generation inside the microfluidic channel with the resultant SERS signal, which is highly reproducible and sensitive. This approach has been used for reproducible detection of protein in the pico to femtomolar concentration. Presented approach is simple, rapid, and cost-effective, and requires low sample volume. Method can be extended for SERS-based detection of other biomolecules.

  12. Development-on-chip: in vitro neural tube patterning with a microfluidic device

    PubMed Central

    Soundararajan, Prabakaran; Chennampally, Phaneendra; Cox, Gregory A.

    2016-01-01

    Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo. PMID:27246712

  13. Patterned electrode-based amperometric gas sensor for direct nitric oxide detection within microfluidic devices.

    PubMed

    Cha, Wansik; Tung, Yi-Chung; Meyerhoff, Mark E; Takayama, Shuichi

    2010-04-15

    This article describes a thin amperometric nitric oxide (NO) sensor that can be microchannel embedded to enable direct real-time detection of NO produced by cells cultured within the microdevice. A key for achieving the thin ( approximately 1 mm) planar sensor configuration required for sensor-channel integration is the use of gold/indium-tin oxide patterned electrode directly on a porous polymer membrane (pAu/ITO) as the base working electrode. The electrochemically deposited Au-hexacyanoferrate layer on pAu/ITO is used to catalyze NO oxidation to nitrite at lower applied potentials (0.65-0.75 V vs Ag/AgCl) and stabilize current output. Furthermore, use of a gas-permeable membrane to separate internal sensor compartments from the sample phase imparts excellent NO selectivity over common interfering agents (e.g., nitrite, ascorbate, ammonia, etc.) present in culture media and biological fluids. The optimized sensor design reversibly detects NO down to the approximately 1 nM level in stirred buffer and <10 nM in flowing buffer when integrated within a polymeric microfluidic device. We demonstrate utility of the channel-embedded sensor by monitoring NO generation from macrophages cultured within non-gas-permeable microchannels, as they are stimulated with endotoxin.

  14. Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis.

    PubMed

    Nguyen, Thi; Kwak, Sukyoung; Karpowicz, Steven J

    2014-11-01

    Microfluidics chip technology is a powerful and convenient alternative to agarose gels and PAGE, but costs can be high due to certain chips being non-reusable. Here we describe a method to regenerate, re-use, and store Agilent DNA, RNA, and protein electrophoresis chips designed for use in the Bioanalyzer 2100. By washing the sample wells and displacing the old gel matrix with new gel-dye mix, we have run samples on the same chip up to ten times with negligible loss of signal quality. Chips whose wells were loaded with buffer or water were stored successfully for one week before re-use.

  15. Protein patterning by a DNA origami framework.

    PubMed

    Aslan, Hüsnü; Krissanaprasit, Abhichart; Besenbacher, Flemming; Gothelf, Kurt V; Dong, Mingdong

    2016-08-18

    A spatial arrangement of proteins provides structural and functional advantages in vast technological applications as well as fundamental research. Most protein patterning procedures employ complicated, time consuming and very costly nanofabrication techniques. As an alternative route, we developed a fully biomolecular self-assembly method using DNA Origami Frames (DOF) as a template for both small and large scale protein patterning. We employed a triangular DOF (tDOF) to arrange the Bovine Serum Albumin (BSA) protein. Our in situ protein patterning strategy provides a novel, fully organic platform using a fast and low-cost surface approach with possible utilization in fundamental science and technological applications.

  16. A double-emulsion microfluidic platform for in vitro green fluorescent protein expression

    NASA Astrophysics Data System (ADS)

    Wu, N.; Oakeshott, J. G.; Easton, C. J.; Peat, T. S.; Surjadi, R.; Zhu, Y.

    2011-05-01

    Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system.

  17. Relationship between functional properties and aggregation changes of whey protein induced by high pressure microfluidization.

    PubMed

    Liu, Cheng-Mei; Zhong, Jun-Zhen; Liu, Wei; Tu, Zong-Cai; Wan, Jie; Cai, Xiao-Fei; Song, Xin-Yun

    2011-05-01

    Aggregation changes of whey protein induced by high-pressure microfluidization (HPM) treatment have been investigated in relation with their functional properties. Whey protein was treated with HPM under pressure from 40 to 160 MPa. Functional properties (solubility, foaming, and emulsifying properties) of whey protein concentrate (WPC) ultrafiltered from fluid whey were evaluated. The results showed significant modifications in the solubility (30% to 59%) and foaming properties (20% to 65%) of WPC with increasing pressure. However, emulsifying property of WPC treated at different pressures was significantly worse than untreated sample. To better understand the mechanism of the modification by HPM, the HPM-induced aggregation changes were examined using particle size distribution, scanning electron microscopy, and hydrophobicity. It was indicated that HPM induced 2 kinds of aggregation changes on WPC: deaggregation and reaggregation of WPC, which resulted in the changes of functional properties of WPC modified by HPM.

  18. Laser-induced fluorescence imaging system for protein separations in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Das, Champak; Stoyanov, Alexander; Fredrickson, Carl; Tran-Son-Tay, Roger; Fan, Zhonghui H.

    2004-12-01

    This paper describes a laser-induced fluorescence (LIF) detection system for imaging proteins separated in a microfluidic device. The diameter of a laser beam is first increased through a beam expander, and subsequently focused into a line using a cylindrical lens. The resultant laser line is used to image an entire capillary or channel in which protein separation took place. The fluorescence emission is collected with a cooled, scientific grade charge-coupled device (CCD) camera. The detection limit was determined using a series of concentrations of fluorescein solutions. The temporal and spatial effects of photobleaching from laser irradiation were analyzed and the parameters to reduce the effect of photobleaching are discussed. We used the imaging system to demonstrate rapid analysis of proteins using isoelectric focusing.

  19. Micropatterning stretched and aligned DNA using microfluidics and surface patterning for applications in hybridization-mediated templated assembly of nanostructures

    NASA Astrophysics Data System (ADS)

    Carbeck, Jeffrey; Petit, Cecilia

    2004-03-01

    Current efforts in nanotechnology use one of two basic approaches: top-down fabrication and bottom-up assembly. Top-down strategies use lithography and contact printing to create patterned surfaces and microfluidic channels that, in turn, can corral and organize nanoscale structures. Bottom-up approaches use templates to direct the assembly of atoms, molecules, and nanoparticles through molecular recognition. The goal of this work is to integrate these strategies by first patterning and orienting DNA molecules through top-down tools so that single DNA chains can then serve as templates for the bottom-up construction of hetero-structures composed of proteins and nanoparticles, both metallic and semi-conducting. The first part of this talk focuses on the top-down strategies used to create microscopic patterns of stretched and aligned molecules of DNA. Specifically, it presents a new method in which molecular combing -- a process by which molecules are deposited and stretched onto a surface by the passage of an air-water interface -- is performed in microchannels. This approach demonstrates that the shape and motion of this interface serve as an effective local field directing the chains dynamically as they are stretched onto the surface. The geometry of the microchannel directs the placement of the DNA molecules, while the geometry of the air-water interface directs the local orientation and curvature of the molecules. This ability to control both the placement and orientation of chains has implication for the use of this technique in genetic analysis and in the bottom up approach to nanofabrication.The second half of this talk presents our bottom-up strategy, which allows placement of nanoparticles along individual DNA chains with a theoretical resolution of less than 1 nm. Specifically, we demonstrate the sequence-specific patterning of nanoparticles via the hybridization of functionalized complementary probes to surface-bound chains of double-stranded DNA. Using

  20. Functionalized poly(ethylene glycol) diacrylate microgels by microfluidics: In situ peptide encapsulation for in serum selective protein detection.

    PubMed

    Celetti, Giorgia; Natale, Concetta Di; Causa, Filippo; Battista, Edmondo; Netti, Paolo A

    2016-09-01

    Polymeric microparticles represent a robustly platform for the detection of clinically relevant analytes in biological samples; they can be functionalized encapsulating a multiple types of biologics entities, enhancing their applications as a new class of colloid materials. Microfluidic offers a versatile platform for the synthesis of monodisperse and engineered microparticles. In this work, we report microfluidic synthesis of novel polymeric microparticles endowed with specific peptide due to its superior specificity for target binding in complex media. A peptide sequence was efficiently encapsulated into the polymeric network and protein binding occurred with high affinity (KD 0.1-0.4μM). Fluidic dynamics simulation was performed to optimize the production conditions for monodisperse and stable functionalized microgels. The results demonstrate the easy and fast realization, in a single step, of functionalized monodisperse microgels using droplet-microfluidic technique, and how the inclusion of the peptide within polymeric network improve both the affinity and the specificity of protein capture.

  1. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

    PubMed Central

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-01-01

    The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  2. Microfluidic devices fabricated using fast wafer-scale LED-lithography patterning.

    PubMed

    Challa, Pavan K; Kartanas, Tadas; Charmet, Jérôme; Knowles, Tuomas P J

    2017-01-01

    Current lithography approaches underpinning the fabrication of microfluidic devices rely on UV exposure of photoresists to define microstructures in these materials. Conventionally, this objective is achieved with gas discharge mercury lamps, which are capable of producing high intensity UV radiation. However, these sources are costly, have a comparatively short lifetime, necessitate regular calibration, and require significant time to warm up prior to exposure taking place. To address these limitations we exploit advances in solid state sources in the UV range and describe a fast and robust wafer-scale laboratory exposure system relying entirely on UV-Light emitting diode (UV-LED) illumination. As an illustration of the potential of this system for fast and low-cost microfluidic device production, we demonstrate the microfabrication of a 3D spray-drying microfluidic device and a 3D double junction microdroplet maker device.

  3. Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices.

    PubMed

    Khnouf, Ruba; Karasneh, Dina; Albiss, Borhan Aldeen

    2016-02-01

    PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most.

  4. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    SciTech Connect

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.

  5. Fabrication of X-ray compatible microfluidic platforms for protein crystallization

    PubMed Central

    Guha, Sudipto; Perry, Sarah L.; Pawate, Ashtamurthy S.; Kenis, Paul J.A.

    2012-01-01

    This paper reports a method for fabricating multilayer microfluidic protein crystallization platforms using different materials to achieve X-ray transparency and compatibility with crystallization reagents. To validate this approach, three soluble proteins, lysozyme, thaumatin, and ribonuclease A were crystallized on-chip, followed by on-chip diffraction data collection. We also report a chip with an array of wells for screening different conditions that consume a minimal amount of protein solution as compared to traditional screening methods. A large number of high quality isomorphous protein crystals can be grown in the wells, after which slices of X-ray data can be collected from many crystals still residing within the wells. Complete protein structures can be obtained by merging these slices of data followed by further processing with crystallography software. This approach of using an x-ray transparent chip for screening, crystal growth, and X-ray data collection enables room temperature data collection from many crystals mounted in parallel, which thus eliminates crystal handling and minimizes radiation damage to the crystals. PMID:23105172

  6. Microfluidic three-dimensional hydrodynamic flow focusing for the rapid protein concentration analysis.

    PubMed

    Hong, Sungmin; Tsou, Pei-Hsiang; Chou, Chao-Kai; Yamaguchi, Hirohito; Su, Chin B; Hung, Mien-Chie; Kameoka, Jun

    2012-06-01

    A simple microfluidic 3D hydrodynamic flow focusing device has been developed and demonstrated quantitative determinations of quantum dot 525 with antibody (QD525-antibody) and hemagglutinin epitope tagged MAX (HA-MAX) protein concentrations. This device had a step depth cross junction structure at a hydrodynamic flow focusing point at which the analyte stream was flowed into a main detection channel and pinched not only horizontally but also vertically by two sheath streams. As a result, a triangular cross-sectional flow profile of the analyte stream was formed and the laser was focused on the top of the triangular shaped analyte stream. Since the detection volume was smaller than the radius of laser spot, a photon burst histogram showed Gaussian distribution, which was necessary for the quantitative analysis of protein concentration. By using this approach, a linear concentration curve of QD525-antibody down to 10 pM was demonstrated. In addition, the concentration of HA-MAX protein in HEK293 cell lysate was determined as 0.283 ± 0.015 nM. This approach requires for only 1 min determining protein concentration. As the best of our knowledge, this is the first time to determinate protein concentration by using single molecule detection techniques.

  7. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    DOE PAGES

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; ...

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

  8. Geometry-induced protein pattern formation.

    PubMed

    Thalmeier, Dominik; Halatek, Jacob; Frey, Erwin

    2016-01-19

    Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in [Formula: see text] EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems.

  9. Sensitive Protein Detection and Quantification in Paper-Based Microfluidics for the Point of Care.

    PubMed

    Anderson, Caitlin E; Shah, Kamal G; Yager, Paul

    2017-01-01

    The design of appropriate diagnostic assays for the point of care requires development of suitable biosensors, detection methods, and diagnostic platforms for sensitive, quantitative detection of biological analytes. Protein targets in particular are especially challenging to detect quantitatively and sensitively due to the lack of amplification strategies akin to nucleic acid amplification. However, recent advances in transducer and biosensor design, new detection labels, and paper-based microfluidics may realize the goal of sensitive, fast, portable, and low-cost protein detection. In this review, we discuss the biochemistry, optics, and engineering advances that may be leveraged to design such a sensitive protein diagnostic assay. The binding kinetics, mechanisms of binding in porous networks, and potential transducers are explained in detail. We discuss the relative merits of various optical detection strategies, potential detection labels, optical readout approaches, and image-processing techniques that are amenable to point-of-care use. To conclude, we present a systematic analysis of potential approaches to enhance the sensitivity of paper-based assays. The assay development framework presented here provides bioassay developers a strategy to methodically enhance the sensitivity and point-of-care suitability of protein diagnostics.

  10. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  11. An off-the-shelf integrated microfluidic device comprising self-assembled monolayers for protein array experiments

    PubMed Central

    Hen, Mirit; Ronen, Maria; Deitch, Alex; Barbiro-Michaely, Efrat; Oren, Ziv; Sukenik, Chaim N.; Gerber, Doron

    2015-01-01

    Microfluidic-based protein arrays are promising tools for life sciences, with increased sensitivity and specificity. One of the drawbacks of this technology is the need to create fresh surface chemistry for protein immobilization at the beginning of each experiment. In this work, we attempted to include the process of surface functionalization as part of the fabrication of the device, which would substitute the time consuming step of surface functionalization at the beginning of each protein array experiment. To this end, we employed a novel surface modification using self-assembled monolayers (SAMs) to immobilize biomolecules within the channels of a polydimethylsiloxane (PDMS) integrated microfluidic device. As a model, we present a general method for depositing siloxane-anchored SAMs, with 1-undecyl-thioacetate-trichlorosilane (C11TA) on the silica surfaces. The process involved developing PDMS-compatible conditions for both SAM deposition and functional group activation. We successfully demonstrated the ability to produce, within an integrated microfluidic channel, a C11TA monolayer with a covalently conjugated antibody. The antibody could then bind its antigen with a high signal to background ratio. We further demonstrated that the antibody was still active after storage of the device for a week. Integration of the surface chemistry into the device as part of its fabrication process has potential to significantly simplify and shorten many experimental procedures involving microfluidic–based protein arrays. In turn, this will allow for broader dissemination of this important technology. PMID:26421087

  12. Protein patterns as endpoints in environmental remediation

    SciTech Connect

    Bradley, B.; Brown, D.

    1995-12-31

    Biological endpoints can complement chemical analyses in monitoring environmental remediation. In some cases the levels of chemical detection are so low that the costs of clean-up to no detection would be prohibitive. And chemical tests do not indicate the availability of the contaminants to the biota. On the other hand many if not most biological tests lack specificity. The authors have investigated a protein expression assay to establish an endpoint for clean-up of sulfur mustard and breakdown products. Earthworms (Lumbricus terrestris) were exposed to sulfur mustard (SM), a breakdown product thiodiethanol (TDE), and ethylene glycol, the solvent for the two chemicals. Tissue from the lining of the coelomic cavity was taken from each of 6 worms in each treatment class. Soluble proteins were extracted and separated on one and two-dimensional (1D and 2D) gels. The 1 D gels showed no difference by eye but the patterns from control and solvent control worms on 2D gels differed from those of worms exposed to TDE and SM. The 1D gel data were digitized and analyzed by pattern recognition using artificial neural networks. The protein patterns under the two treatments and the two controls were learned in one set of data and successfully recognized in a second. This indicated that what was learned was useful in recognizing patterns induced by SM and TDE. Thus a possible endpoint for remediation would be the protein pattern at no effect levels of chemicals of interest.

  13. Fabrication of a polystyrene microfluidic chip coupled to electrospray ionization mass spectrometry for protein analysis.

    PubMed

    Hu, Xianqiao; Dong, Yuanyuan; He, Qiaohong; Chen, Hengwu; Zhu, Zhiwei

    2015-05-15

    A highly integrated polystyrene (PS) microfluidic chip coupled to electrospray ionization mass spectrometry for on-chip protein digestion and online analysis was developed. The immobilized enzymatic microreactor for on-chip protein digestion was integrated onto microchip via the novel method of region-selective UV-modification combined with glutaraldehyde-based immobilization. The micro film electric contact for applying high voltage was prepared on chips by using UV-directed electroless plating technique. A micro-tip was machined at the end of main channel, serving as the interface between microchip and mass spectrometric detector. On-chip digestion and online detection of protein was carried out by coupling the microchip with mass spectrometry (MS). The influences of methanol flow rate in side channel on the stability of spray and intensity of signals were investigated systematically. Also the influence of sample flow rate on the performance of immobilized enzymatic reactor were investigated. Stable spray was obtained at the spray voltage of 2.8-3.0kV and the methanol flow rate of 500-700nLmin(-1) with the relative standard deviation (RSD) of total ion current (TIC) less than 10%. The influence of sample flow rate on the performance of immobilized enzymatic reactor was also studied. The sequence coverage of protein identification decreased with the increase of flow rate of the sample solution. A sequence coverage of 96% was obtained with immobilized enzymatic reactor at the sample flow rate of 100nLmin(-1) with the reaction time of 8.4min. It could detect cytochrome c as low as 10μgmL(-1) with the developed system. No obvious decrease in protein digestion efficiency was observed after the chip continuously performed for 4h and stored for 15d.

  14. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    SciTech Connect

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method will also

  15. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    DOE PAGES

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; ...

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles canmore » be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method

  16. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    PubMed Central

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-01-01

    The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis

  17. Different migration patterns of sea urchin and mouse sperm revealed by a microfluidic chemotaxis device.

    PubMed

    Chang, Haixin; Kim, Beum Jun; Kim, Yoon Soo; Suarez, Susan S; Wu, Mingming

    2013-01-01

    Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin Arbacia punctulata sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity Vx up the chemical gradient as high as 20% of its average speed (238 μm/s), while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.

  18. QR-on-a-chip: a computer-recognizable micro-pattern engraved microfluidic device for high-throughput image acquisition.

    PubMed

    Yun, Kyungwon; Lee, Hyunjae; Bang, Hyunwoo; Jeon, Noo Li

    2016-02-21

    This study proposes a novel way to achieve high-throughput image acquisition based on a computer-recognizable micro-pattern implemented on a microfluidic device. We integrated the QR code, a two-dimensional barcode system, onto the microfluidic device to simplify imaging of multiple ROIs (regions of interest). A standard QR code pattern was modified to arrays of cylindrical structures of polydimethylsiloxane (PDMS). Utilizing the recognition of the micro-pattern, the proposed system enables: (1) device identification, which allows referencing additional information of the device, such as device imaging sequences or the ROIs and (2) composing a coordinate system for an arbitrarily located microfluidic device with respect to the stage. Based on these functionalities, the proposed method performs one-step high-throughput imaging for data acquisition in microfluidic devices without further manual exploration and locating of the desired ROIs. In our experience, the proposed method significantly reduced the time for the preparation of an acquisition. We expect that the method will innovatively improve the prototype device data acquisition and analysis.

  19. Enhanced detection of quantum dots labeled protein by simultaneous bismuth electrodeposition into microfluidic channel.

    PubMed

    Medina-Sánchez, Mariana; Miserere, Sandrine; Cadevall, Miquell; Merkoçi, Arben

    2016-02-01

    In this study, we propose an electrochemical immunoassay into a disposable microfluidic platform, using quantum dots (QDs) as labels and their enhanced detection using bismuth as an alternative to mercury electrodes. CdSe@ZnS QDs were used to tag human IgG as a model protein and detected through highly sensitive stripping voltammetry of the dissolved metallic component (cadmium in our case). The modification of the screen printed carbon electrodes (SPCEs) was done by a simple electrodeposition of bismuth that was previously mixed with the sample containing QDs. A magneto-immunosandwich assay was performed using a micromixer. A magnet placed at its outlet in order to capture the magnetic beads used as solid support for the immunoassay. SPCEs were integrated at the end of the channel as detector. Different parameters such as bismuth concentration, flow rate, and incubation times, were optimized. The LOD for HIgG in presence of bismuth was 3.5 ng/mL with a RSD of 13.2%. This LOD was about 3.3-fold lower than the one obtained without bismuth. Furthermore, the sensitivity of the system was increased 100-fold respect to experiments carried out with classical screen-printed electrodes, both in presence of bismuth.

  20. Hybrid Magnetic-DNA Directed Immobilisation Approach for Efficient Protein Capture and Detection on Microfluidic Platforms.

    PubMed

    Esmaeili, Elaheh; Ghiass, Mohammad Adel; Vossoughi, Manouchehr; Soleimani, Masoud

    2017-03-15

    In this study, a hybrid magnetic-DNA directed immobilisation approach is presented to enhance protein capture and detection on a microfluidic platform. DNA-modified magnetic nanoparticles are added in a solution to capture fluorescently labelled immunocomplexes to be detected optically. A magnetic set-up composed of cubic permanent magnets and a microchannel was designed and implemented based on finite element analysis results to efficiently concentrate the nanoparticles only over a defined area of the microchannel as the sensing zone. This in turn, led to the fluorescence emission localisation and the searching area reduction. Also, compared to processes in which the immunocomplex is formed directly on the surface, the proposed approach provides a lower steric hindrance, higher mass transfer, lower equilibrium time, and more surface concentration of the captured targets leading to a faster and more sensitive detection. As a proof-of-concept, the set-up is capable of detecting prostate-specific membrane antigen with concentrations down to 0.7 nM. Our findings suggest that the approach holds a great promise for applications in clinical assays and disease diagnosis.

  1. Using Three-Phase Flow of Immiscible Liquids to Prevent Coalescence of Droplets in Microfluidic Channels: Criteria to Identify theThird Liquid and Validation with Protein Crystallizations

    SciTech Connect

    Chen, D.; Li, L; Reyes, S; Adamson, D; Ismagilov, R

    2007-01-01

    This manuscript describes the effect of interfacial tensions on three-phase liquid-liquid-liquid flow in microfluidic channels and the use of this flow to prevent microfluidic plugs from coalescing. One problem in using microfluidic plugs as microreactors is the coalescence of adjacent plugs caused by the relative motion of plugs during flow. Here, coalescence of reagent plugs was eliminated by using plugs of a third immiscible liquid as spacers to separate adjacent reagent plugs. This work tested the requirements of interfacial tensions for plugs of a third liquid to be effective spacers. Two candidates satisfying the requirements were identified, and one of these liquids was used in the crystallization of protein human Tdp1 to demonstrate its compatibility with protein crystallization in plugs. This method for identifying immiscible liquids for use as a spacer will also be useful for applications involving manipulation of large arrays of droplets in microfluidic channels.

  2. Ultrasensitive detection of low-abundance surface-marker protein using isothermal rolling circle amplification in a microfluidic nanoliter platform.

    PubMed

    Konry, Tania; Smolina, Irina; Yarmush, Joel M; Irimia, Daniel; Yarmush, Martin L

    2011-02-07

    With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection events that can be greatly amplified via isothermal rolling circle amplification (RCA) is applied. By merging the single-molecule detection power of RCA reactions with microfluidic technology, it is demonstrated that the identification of specific protein markers can be achieved on tumor-cell surfaces in miniaturized nanoliter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer.

  3. Finding protein-protein interaction patterns by contact map matching.

    PubMed

    Melo, R C; Ribeiro, C; Murray, C S; Veloso, C J M; da Silveira, C H; Neshich, G; Meira, W; Carceroni, R L; Santoro, M M

    2007-10-05

    We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature.

  4. Coupling of a microfluidic mixer to a Fourier-transform infrared spectrometer for protein-conformation studies.

    PubMed

    Prim, Denis; Crelier, Simon; Segura, Jean-Manuel

    2011-01-01

    The biological properties of a protein critically depend on its conformation, which can vary as a result of changes in conditions such as pH or following the addition of various substances. Being able to reliably assess the quality of protein structures under various conditions is therefore of crucial importance. Infrared (IR) spectroscopy of the Amide I band of proteins is a powerful method for the determination of protein conformations and further allows the analysis of continuously flowing solutions of the target molecule. Here, a commercial Fourier-transform infrared spectrometer was coupled to a microfluidic mixer to allow the on-line monitoring of protein conformation under varying conditions. The validity of the concept was demonstrated by continuously recording the variations of the IR spectrum of poly-L-lysine resulting from repetitive, pH-induced conformational changes.

  5. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  6. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.

    PubMed

    Blackburn, Matthew C; Petrova, Ekaterina; Correia, Bruno E; Maerkl, Sebastian J

    2016-04-20

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

  7. A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

    PubMed

    Soroush, Fariborz; Zhang, Ting; King, Devon J; Tang, Yuan; Deosarkar, Sudhir; Prabhakarpandian, Balabhaskar; Kilpatrick, Laurie E; Kiani, Mohammad F

    2016-11-01

    A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

  8. Technical Advance: Changes in neutrophil migration patterns upon contact with platelets in a microfluidic assay.

    PubMed

    Frydman, Galit H; Le, Anna; Ellett, Felix; Jorgensen, Julianne; Fox, James G; Tompkins, Ronald G; Irimia, Daniel

    2017-03-01

    Neutrophils are traditionally regarded as the "first responders" of the immune system. However, recent observations revealed that platelets often respond earlier to recruit and activate neutrophils within sites of injury and inflammation. Currently, platelet-neutrophil interactions are studied by intravital microscopy. Although such studies provide exceptional, physiologic in vivo data, they are also laborious and have low throughput. To accelerate platelet-neutrophil interaction studies, we have developed and optimized an ex vivo microfluidic platform with which the interactions between platelets and moving neutrophils are measured at single-cell level in precise conditions and with high throughput. With the use of this new assay, we have evaluated changes in neutrophil motility upon direct contact with platelets. Motility changes include longer distances traveled, frequent changes in direction, and faster neutrophil velocities compared with a standard motility response to chemoattractant fMLP. We also found that the neutrophil-platelet direct interactions are transient and mediated by CD62P-CD162 interactions, localized predominantly at the uropod of moving neutrophils. This "crawling," oscillatory neutrophil behavior upon platelet contact is consistent with previous in vivo studies and validates the use of this new test for the exploration of this interactive relationship.

  9. Microfluidics and Coagulation Biology

    PubMed Central

    Colace, Thomas V.; Tormoen, Garth W.

    2014-01-01

    The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices facilitate measurements of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, pharmacology, and clinical diagnostics. An experimental session can accommodate 100s to 1000s of unique clotting events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor under constant flow rate or constant pressure drop conditions. Distinct shear rates can be created on a device with a single perfusion pump. Microfluidic devices facilitated the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidics are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to the mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics. PMID:23642241

  10. Coupling High Throughput Microfluidics and Small-Angle X-ray Scattering to Study Protein Crystallization from Solution.

    PubMed

    Pham, Nhat; Radajewski, Dimitri; Round, Adam; Brennich, Martha; Pernot, Petra; Biscans, Béatrice; Bonneté, Françoise; Teychené, Sébastien

    2017-02-21

    In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams.

  11. Sedimentation Patterns of Rapidly Reversible Protein Interactions

    PubMed Central

    Schuck, Peter

    2010-01-01

    Abstract The transport behavior of macromolecular mixtures with rapidly reversible complex formation is of great interest in the study of protein interactions by many different methods. Complicated transport patterns arise even for simple bimolecular reactions, when all species exhibit different migration velocities. Although partial differential equations are available to describe the spatial and temporal evolution of the interacting system given particular initial conditions, a general overview of the phase behavior of the systems in parameter space has not yet been reported. In the case of sedimentation of two-component mixtures, this study presents simple analytical solutions that solve the underlying equations in the diffusion-free limit previously subject to Gilbert-Jenkins theory. The new expressions describe, with high precision, the average sedimentation coefficients and composition of each boundary, which allow the examination of features of the whole parameter space at once, and may be used for experimental design and robust analysis of experimental boundary patterns to derive the stoichiometry and affinity of the complex. This study finds previously unrecognized features, including a phase transition between boundary patterns. The model reveals that the time-average velocities of all components in the reaction mixture must match—a condition that suggests an intuitive physical picture of an effective particle of the coupled cosedimentation of an interacting system. Adding to the existing numerical solutions of the relevant partial differential equations, the effective particle model provides physical insights into the relationships of the parameters that govern sedimentation patterns. PMID:20441765

  12. DETECTION OF TOPOLOGICAL PATTERNS IN PROTEIN NETWORKS.

    SciTech Connect

    MASLOV,S.SNEPPEN,K.

    2003-11-17

    property of many biological networks that was recently brought to attention of the scientific community [3, 4, 5] is an extremely broad distribution of node connectivities defined as the number of immediate neighbors of a given node in the network. While the majority of nodes have just a few edges connecting them to other nodes in the network, there exist some nodes, that we will refer to as ''hubs'', with an unusually large number of neighbors. The connectivity of the most connected hub in such a network is typically several orders of magnitude larger than the average connectivity in the network. Often the distribution of connectivities of individual nodes can be approximated by a scale-free power law form [3] in which case the network is referred to as scale-free. Among biological networks distributions of node connectivities in metabolic [4], protein interaction [5], and brain functional [6] networks can be reasonably approximated by a power law extending for several orders of magnitude. The set of connectivities of individual nodes is an example of a low-level (single-node) topological property of a network. While it answers the question about how many neighbors a given node has, it gives no information about the identity of those neighbors. It is clear that most functional properties of networks are defined at a higher topological level in the exact pattern of connections of nodes to each other. However, such multi-node connectivity patterns are rather difficult to quantify and compare between networks. In this work we concentrate on multi-node topological properties of protein networks. These networks (as any other biological networks) lack the top-down design. Instead, selective forces of biological evolution shape them from raw material provided by random events such as mutations within individual genes, and gene duplications. As a result their connections are characterized by a large degree of randomness. One may wonder which connectivity patterns are indeed

  13. Versatile multiple protein nanopatterning within a microfluidic channel for cell recruitment studies.

    PubMed

    Andersen, A S; Zheng, W F; Sutherland, D S; Jiang, X Y

    2015-12-21

    A novel approach combining self-assembly-based colloidal lithography and polydimethylsiloxane (PDMS) micromolding to generate complex protein nanopatterns for studying the mechanisms of leukocyte extravasation within microchannels is presented. Nanostructured surfaces sealed onto PDMS-molded microchannels are chemically functionalized in situ in an all-aqueous process to generate bi-functional chemical nanopatterns. Subsequent co-immobilization with proteins makes use of common non-covalent coupling (e.g. HIS-tags, FC-tags and biotin-tags), giving nanopatterns of arbitrary combinations of oriented, functional proteins. Up to three different proteins were simultaneously co-immobilized into the microchannel with nanoscale precision, demonstrating the complex patterns. As a proof-of-principle, a mimic of an inflamed endothelium was constructed using a macro- and nanoscale pattern of intercellular adhesion molecule 1 (ICAM1) and P-selectin, and the response of leukocytes through live cell imaging was measured. A clear result on the rolling behavior of the cells was observed with rolling limited to areas where ICAM1 and P-selectin are present. This micro/nano-interface will open new doors to investigations of how spatial distributions of proteins control cellular activity.

  14. Magnetolithographic patterning of inner walls of a tube: a new dimension in microfluidics and sequential microreactors.

    PubMed

    Bardea, Amos; Baram, Aviad; Tatikonda, Anand Kumar; Naaman, Ron

    2009-12-30

    By applying magnetolithography it is possible to chemically pattern the inside of tubes. This new capability allows one to perform sequential processes within the tubes. Several enzymatic reactions are demonstrated.

  15. A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract.

    PubMed

    Wu, N; Zhu, Y; Brown, S; Oakeshott, J; Peat, T S; Surjadi, R; Easton, C; Leech, P W; Sexton, B A

    2009-12-07

    Droplet based microfluidics are promising new tools for biological and chemical assays. In this paper, a high throughput and high sensitivity microfluidic droplet platform is described for in vitro protein expression using crude Escherichia coli S30 extract. A flow-focusing polymethylmethacrylate (PMMA) microchip was designed and integrated with different functions involving droplet generation, storage, separation and detection. The material used for the chip is superior to the previously tested polydimethylsiloxane (PDMS) due to its mechanical and chemical properties. Droplet formation characteristics such as size and generation rate are investigated systematically. The effect of surfactants Abil EM90 and Span80 in the oil phase on droplet formation and optical detection is also studied. The performance of the system is demonstrated by the high throughput and stable droplet generation and ultralow detection limit. The robustness of the system is also demonstrated by the successful synthesis of a green fluorescent protein (GFP) using E. coli S30 extract as a source of RNA translation reagents.

  16. Microfluidic device, and related methods

    NASA Technical Reports Server (NTRS)

    Wong, Eric W. (Inventor)

    2010-01-01

    A method of making a microfluidic device is provided. The method features patterning a permeable wall on a substrate, and surrounding the permeable wall with a solid, non-permeable boundary structure to establish a microfluidic channel having a cross-sectional dimension less than 5,000 microns and a cross-sectional area at least partially filled with the permeable wall so that fluid flowing through the microfluidic channel at least partially passes through the permeable wall.

  17. Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

    PubMed Central

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-01-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells. PMID:24938227

  18. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  19. Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

    PubMed

    Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

    2016-05-15

    In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future.

  20. Suspended microfluidics.

    PubMed

    Casavant, Benjamin P; Berthier, Erwin; Theberge, Ashleigh B; Berthier, Jean; Montanez-Sauri, Sara I; Bischel, Lauren L; Brakke, Kenneth; Hedman, Curtis J; Bushman, Wade; Keller, Nancy P; Beebe, David J

    2013-06-18

    Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

  1. A critical insight into the development pipeline of microfluidic immunoassay devices for the sensitive quantitation of protein biomarkers at the point of care.

    PubMed

    Barbosa, Ana I; Reis, Nuno M

    2017-03-13

    The latest clinical procedures for the timely and cost-effective diagnosis of chronic and acute clinical conditions, such as cardiovascular diseases, cancer, chronic respiratory diseases, diabetes or sepsis (i.e. the biggest causes of death worldwide), involve the quantitation of specific protein biomarkers released into the blood stream or other physiological fluids (e.g. urine or saliva). The clinical thresholds are usually in the femtomolar to picolomar range, and consequently the measurement of these protein biomarkers heavily relies on highly sophisticated, bulky and automated equipment in centralised pathology laboratories. The first microfluidic devices capable of measuring protein biomarkers in miniaturised immunoassays were presented nearly two decades ago and promised to revolutionise point-of-care (POC) testing by offering unmatched sensitivity and automation in a compact POC format; however, the development and adoption of microfluidic protein biomarker tests has fallen behind expectations. This review presents a detailed critical overview into the pipeline of microfluidic devices developed in the period 2005-2016 capable of measuring protein biomarkers from the pM to fM range in formats compatible with POC testing, with a particular focus on the use of affordable microfluidic materials and compact low-cost signal interrogation. The integration of these two important features (essential unique selling points for the successful microfluidic diagnostic products) has been missed in previous review articles and explain the poor adoption of microfluidic technologies in this field. Most current miniaturised devices compromise either on the affordability, compactness and/or performance of the test, making current tests unsuitable for the POC measurement of protein biomarkers. Seven core technical areas, including (i) the selected strategy for antibody immobilisation, (ii) the surface area and surface-area-to-volume ratio, (iii) surface passivation, (iv) the

  2. New developments in protein structure-function analysis by MS and use of hydrogen-deuterium exchange microfluidics.

    PubMed

    Landreh, Michael; Astorga-Wells, Juan; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-10-01

    The study of protein structure and function has evolved to become a leading discipline in the biophysical sciences. Although it is not yet possible to determine 3D protein structures from MS data alone, multiple MS-based techniques can be combined to obtain structural and functional data that are complementary to classical protein structure information obtained from NMR or X-ray crystallography. Monitoring gas-phase interactions of noncovalent complexes yields information on binding constants, complex stability, and the nature of interactions. Ion mobility MS and chemical crosslinking strategies can be applied to probe the architecture of macromolecular assemblies and protein-ligand complexes. MS analysis of hydrogen-deuterium exchange can be used to determine the localization of secondary structure elements, binding sites and conformational dynamics of proteins in solution. This minireview focuses first on new strategies that combine these techniques to gain insights into protein structure and function. Using one such strategy, we then demonstrate how a novel hydrogen-deuterium exchange microfluidics tool can be used online with an ESI mass spectrometer to monitor regional accessibility in a peptide, as exemplified with amyloid-β peptide 1-40.

  3. A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots.

    PubMed

    Wouters, Bert; Dapic, Irena; Valkenburg, Thalassa S E; Wouters, Sam; Niezen, Leon; Eeltink, Sebastiaan; Corthals, Garry L; Schoenmakers, Peter J

    2017-03-31

    A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character.

  4. Automatic disease screening method using image processing for dried blood microfluidic drop stain pattern recognition.

    PubMed

    Sikarwar, Basant S; Roy, Mukesh; Ranjan, Priya; Goyal, Ayush

    2016-07-01

    This paper examines programmed automatic recognition of infection from samples of dried stains of micro-scale drops of patient blood. This technique has the upside of being low-cost and less-intrusive and not requiring puncturing the patient with a needle for drawing blood, which is especially critical for infants and the matured. It also does not require expensive pathological blood test laboratory equipment. The method is shown in this work to be successful for ailment identification in patients suffering from tuberculosis and anaemia. Illness affects the physical properties of blood, which thus influence the samples of dried micro-scale blood drop stains. For instance, if a patient has a severe drop in platelet count, which is often the case of dengue or malaria patients, the blood's physical property of viscosity drops substantially, i.e. the blood is thinner. Thus, the blood micro-scale drop stain samples can be utilised for diagnosing maladies. This paper presents programmed automatic examination of the dried micro-scale drop blood stain designs utilising an algorithm based on pattern recognition. The samples of micro-scale blood drop stains of ordinary non-infected people are clearly recognisable as well as the samples of micro-scale blood drop stains of sick people, due to key distinguishing features. As a contextual analysis, the micro-scale blood drop stains of patients infected with tuberculosis have been contrasted with the micro-scale blood drop stains of typical normal healthy people. The paper dives into the fundamental flow mechanics behind how the samples of the dried micro-scale blood drop stain is shaped. What has been found is a thick ring like feature in the dried micro-scale blood drop stains of non-ailing people and thin shape like lines in the dried micro-scale blood drop stains of patients with anaemia or tuberculosis disease. The ring like feature at the periphery is caused by an outward stream conveying suspended particles to the edge

  5. Simple Host—Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    PubMed Central

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2008-01-01

    This paper utilizes cyclodextrin-based host—guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-β-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way. PMID:18831551

  6. Simple Host−Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    SciTech Connect

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2009-01-15

    This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-{beta}-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

  7. Microfluidic electronics.

    PubMed

    Cheng, Shi; Wu, Zhigang

    2012-08-21

    Microfluidics, a field that has been well-established for several decades, has seen extensive applications in the areas of biology, chemistry, and medicine. However, it might be very hard to imagine how such soft microfluidic devices would be used in other areas, such as electronics, in which stiff, solid metals, insulators, and semiconductors have previously dominated. Very recently, things have radically changed. Taking advantage of native properties of microfluidics, advances in microfluidics-based electronics have shown great potential in numerous new appealing applications, e.g. bio-inspired devices, body-worn healthcare and medical sensing systems, and ergonomic units, in which conventional rigid, bulky electronics are facing insurmountable obstacles to fulfil the demand on comfortable user experience. Not only would the birth of microfluidic electronics contribute to both the microfluidics and electronics fields, but it may also shape the future of our daily life. Nevertheless, microfluidic electronics are still at a very early stage, and significant efforts in research and development are needed to advance this emerging field. The intention of this article is to review recent research outcomes in the field of microfluidic electronics, and address current technical challenges and issues. The outlook of future development in microfluidic electronic devices and systems, as well as new fabrication techniques, is also discussed. Moreover, the authors would like to inspire both the microfluidics and electronics communities to further exploit this newly-established field.

  8. Surface acoustic wave microfluidics.

    PubMed

    Ding, Xiaoyun; Li, Peng; Lin, Sz-Chin Steven; Stratton, Zackary S; Nama, Nitesh; Guo, Feng; Slotcavage, Daniel; Mao, Xiaole; Shi, Jinjie; Costanzo, Francesco; Huang, Tony Jun

    2013-09-21

    The recent introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip platforms has opened a new frontier in microfluidics. The advantages provided by such SAW microfluidics are numerous: simple fabrication, high biocompatibility, fast fluid actuation, versatility, compact and inexpensive devices and accessories, contact-free particle manipulation, and compatibility with other microfluidic components. We believe that these advantages enable SAW microfluidics to play a significant role in a variety of applications in biology, chemistry, engineering and medicine. In this review article, we discuss the theory underpinning SAWs and their interactions with particles and the contacting fluids in which they are suspended. We then review the SAW-enabled microfluidic devices demonstrated to date, starting with devices that accomplish fluid mixing and transport through the use of travelling SAW; we follow that by reviewing the more recent innovations achieved with standing SAW that enable such actions as particle/cell focusing, sorting and patterning. Finally, we look forward and appraise where the discipline of SAW microfluidics could go next.

  9. Surface acoustic wave microfluidics

    PubMed Central

    Ding, Xiaoyun; Li, Peng; Lin, Sz-Chin Steven; Stratton, Zackary S.; Nama, Nitesh; Guo, Feng; Slotcavage, Daniel; Mao, Xiaole; Shi, Jinjie; Costanzo, Francesco; Huang, Tony Jun

    2014-01-01

    The recent introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip platforms has opened a new frontier in microfluidics. The advantages provided by such SAW microfluidics are numerous: simple fabrication, high biocompatibility, fast fluid actuation, versatility, compact and inexpensive devices and accessories, contact-free particle manipulation, and compatibility with other microfluidic components. We believe that these advantages enable SAW microfluidics to play a significant role in a variety of applications in biology, chemistry, engineering, and medicine. In this review article, we discuss the theory underpinning SAWs and their interactions with particles and the contacting fluids in which they are suspended. We then review the SAW-enabled microfluidic devices demonstrated to date, starting with devices that accomplish fluid mixing and transport through the use of travelling SAW; we follow that by reviewing the more recent innovations achieved with standing SAW that enable such actions as particle/cell focusing, sorting, and patterning. Finally, we look forward and appraise where the discipline of SAW microfluidics could go next. PMID:23900527

  10. Electro-Microfluidic Packaging

    SciTech Connect

    BENAVIDES, GILBERT L.; GALAMBOS, PAUL C.

    2002-06-01

    Electro-microfluidics is experiencing explosive growth in new product developments. There are many commercial applications for electro-microfluidic devices such as chemical sensors, biological sensors, and drop ejectors for both printing and chemical analysis. The number of silicon surface micromachined electro-microfluidic products is likely to increase. Manufacturing efficiency and integration of microfluidics with electronics will become important. Surface micromachined microfluidic devices are manufactured with the same tools as IC's (integrated circuits) and their fabrication can be incorporated into the IC fabrication process. In order to realize applications for devices must be developed. An Electro-Microfluidic Dual In-line Package (EMDIP{trademark}) was developed surface micromachined electro-microfluidic devices, a practical method for getting fluid into these to be a standard solution that allows for both the electrical and the fluidic connections needed to operate a great variety of electro-microfluidic devices. The EMDIP{trademark} includes a fan-out manifold that, on one side, mates directly with the 200 micron diameter Bosch etched holes found on the device, and, on the other side, mates to lager 1 mm diameter holes. To minimize cost the EMDIP{trademark} can be injection molded in a great variety of thermoplastics which also serve to optimize fluid compatibility. The EMDIP{trademark} plugs directly into a fluidic printed wiring board using a standard dual in-line package pattern for the electrical connections and having a grid of multiple 1 mm diameter fluidic connections to mate to the underside of the EMDIP{trademark}.

  11. Control of crystal polymorph in microfluidics using molluscan 28 kDa Ca²(+)-binding protein.

    PubMed

    Ji, Bozhi; Cusack, Maggie; Freer, Andy; Dobson, Phil S; Gadegaard, Nikolaj; Yin, Huabing

    2010-10-01

    Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery.

  12. Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.

    PubMed

    Arosio, Paolo; Müller, Thomas; Rajah, Luke; Yates, Emma V; Aprile, Francesco A; Zhang, Yingbo; Cohen, Samuel I A; White, Duncan A; Herling, Therese W; De Genst, Erwin J; Linse, Sara; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2016-01-26

    Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.

  13. PDMS bonding to a bio-friendly photoresist via self-polymerized poly(dopamine) adhesive for complex protein micropatterning inside microfluidic channels.

    PubMed

    Kim, Miju; Song, Kwang Hoon; Doh, Junsang

    2013-12-01

    Protein micropatterned surfaces integrated with microfluidics are useful in numerous bioanalytical and biological applications. In this study, we demonstrated the fabrication of complex protein micropatterned surfaces within poly(dimethylsiloxane) (PDMS) microfluidic channels by attaching the PDMS channels to bio-friendly photoresist films and subsequently performing microscope projection photolithography (MPP). A muscle-inspired poly(dopamine) (PDA) coating was employed to mediate the bonding between the PDMS and the bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP). By adjusting the dip-coating time for the PDA coating, we could successfully introduce sufficient amounts of functional groups on the PDMP surfaces to mediate strong bonding between the PDMS channels and the PDA-coated PDMP thin films with minimal alteration of the surface properties of the PDMP thin films that are critical for protein micropatterning. Using this novel bonding strategy, we successfully fabricated multiple protein micropatterns and gradient micropatterns of proteins within microfluidic channels. The technique developed in this study will be useful for the fabrication of complex biochips for multiplex bioassays and fundamental cell biological studies.

  14. Microfluidics experiments of dissolution in a fracture. Influence of Damköhler and Péclet numbers, and of the geometry on the dissolution pattern

    NASA Astrophysics Data System (ADS)

    Osselin, Florian; Budek, Agnieszka; Cybulski, Olgierd; Szymczak, Piotr

    2015-04-01

    Dissolution of natural rocks is an ever present phenomenon in nature. The shaping of natural landscapes by the dissolution of limestone gives for example birth to exceptional features like karsts. Currently dissolution is also at the heart of key research topics as Carbon Capture and Storage or Enhanced Oil Recovery. The basics principles of dissolution are well-known, however, the sheer amount of different patterns arising from these mechanisms and the strong dependency on parameters such as pore network, chemical composition and flow rate, make it particularly difficult to study theoretically and experimentally. In this study we present a microfluidic experiment simulating the behavior of a dissolving fluid in a fracture. The experiments consist of a chip of gyspum inserted between two polycarbonate plates and subjected to a constant flow rate of pure water. The point in using microfluidics is that it allows a complete control on the experimental parameters such as geometry and chemical composition of the porous medium, flow rate, fracture aperture, roughness of the fracture walls, and an in situ observation of the geometry evolution which is impossible with 3D natural rocks. Thanks to our experiments we have been able to cover the whole range of dissolution patterns, from wormholing or DLA fingering to homogeneous dissolution, by changing Péclet and Damköhler numbers. Moreover, we have been able to tweak the geometry of our artificial fracture, inserting finger seeds or non-dissolvable obstacles. The comparison of the experimental patterns with the numerical dissolution code dissol (Szymczak and Ladd 2011) has then shown a very good correlation of the patterns, giving confidence in both experiments and modeling.

  15. Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry.

    PubMed

    Rob, Tamanna; Liuni, Peter; Gill, Preet Kamal; Zhu, Shaolong; Balachandran, Naresh; Berti, Paul J; Wilson, Derek J

    2012-04-17

    This work introduces an integrated microfluidic device for measuring rapid H/D exchange (HDX) in proteins. By monitoring backbone amide HDX on the millisecond to low second time scale, we are able to characterize conformational dynamics in weakly structured regions, such as loops and molten globule-like domains that are inaccessible in conventional HDX experiments. The device accommodates the entire MS-based HDX workflow on a single chip with residence times sufficiently small (ca. 8 s) that back-exchange is negligible (≤5%), even without cooling. Components include an adjustable position capillary mixer providing a variable-time labeling pulse, a static mixer for HDX quenching, a proteolytic microreactor for rapid protein digestion, and on-chip electrospray ionization (ESI). In the present work, we characterize device performance using three model systems, each illustrating a different application of 'time-resolved' HDX. Ubiquitin is used to illustrate a crude, high throughput structural analysis based on a single subsecond HDX time-point. In experiments using cytochrome c, we distinguish dynamic behavior in loops, establishing a link between flexibility and interactions with the heme prosthetic group. Finally, we localize an unusually high 'burst-phase' of HDX in the large tetrameric enzyme DAHP synthase to a 'molten globule-like' region surrounding the active site.

  16. A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

    PubMed

    Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

    2013-08-21

    Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

  17. Liquid metal enabled microfluidics.

    PubMed

    Khoshmanesh, Khashayar; Tang, Shi-Yang; Zhu, Jiu Yang; Schaefer, Samira; Mitchell, Arnan; Kalantar-Zadeh, Kourosh; Dickey, Michael D

    2017-03-14

    Several gallium-based liquid metal alloys are liquid at room temperature. As 'liquid', such alloys have a low viscosity and a high surface tension while as 'metal', they have high thermal and electrical conductivities, similar to mercury. However, unlike mercury, these liquid metal alloys have low toxicity and a negligible vapor pressure, rendering them much safer. In comparison to mercury, the distinguishing feature of these alloys is the rapid formation of a self-limiting atomically thin layer of gallium oxide over their surface when exposed to oxygen. This oxide layer changes many physical and chemical properties of gallium alloys, including their interfacial and rheological properties, which can be employed and modulated for various applications in microfluidics. Injecting liquid metal into microfluidic structures has been extensively used to pattern and encapsulate highly deformable and reconfigurable electronic devices including electrodes, sensors, antennas, and interconnects. Likewise, the unique features of liquid metals have been employed for fabricating miniaturized microfluidic components including pumps, valves, heaters, and electrodes. In this review, we discuss liquid metal enabled microfluidic components, and highlight their desirable attributes including simple fabrication, facile integration, stretchability, reconfigurability, and low power consumption, with promising applications for highly integrated microfluidic systems.

  18. Adsorption of HP Lattice Proteins on Patterned Surfaces

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew; Shi, Guangjie; Landau, David P.; Li, Ying Wai; Wuest, Thomas

    2014-03-01

    The HP lattice model[2] is a course-grained, yet useful tool for modeling protein sequences where amino acids are treated as either hydrophobic (H) or polar (P) monomers. With the use of Wang-Landau sampling and an efficient set of Monte-Carlo moves[3], HP lattice proteins adsorbed on patterned surfaces are studied. Each substrate is modeled as a periodically bounded pattern of lattice sites that interact with either H or P monomers in the lattice protein, where the energy contributions of the surface are determined by assigned coupling strengths. By analyzing energy degeneracies, along with the thermodynamic and structural quantities of the protein, both the protein folding and surface adsorption can be observed. The adsorption behavior of the lattice proteins on patterned surfaces will be compared to those interacting with uniform surfaces. Research supported by NSF.

  19. Encoding protein-ligand interaction patterns in fingerprints and graphs.

    PubMed

    Desaphy, Jérémy; Raimbaud, Eric; Ducrot, Pierre; Rognan, Didier

    2013-03-25

    We herewith present a novel and universal method to convert protein-ligand coordinates into a simple fingerprint of 210 integers registering the corresponding molecular interaction pattern. Each interaction (hydrophobic, aromatic, hydrogen bond, ionic bond, metal complexation) is detected on the fly and physically described by a pseudoatom centered either on the interacting ligand atom, the interacting protein atom, or the geometric center of both interacting atoms. Counting all possible triplets of interaction pseudoatoms within six distance ranges, and pruning the full integer vector to keep the most frequent triplets enables the definition of a simple (210 integers) and coordinate frame-invariant interaction pattern descriptor (TIFP) that can be applied to compare any pair of protein-ligand complexes. TIFP fingerprints have been calculated for ca. 10,000 druggable protein-ligand complexes therefore enabling a wide comparison of relationships between interaction pattern similarity and ligand or binding site pairwise similarity. We notably show that interaction pattern similarity strongly depends on binding site similarity. In addition to the TIFP fingerprint which registers intermolecular interactions between a ligand and its target protein, we developed two tools (Ishape, Grim) to align protein-ligand complexes from their interaction patterns. Ishape is based on the overlap of interaction pseudoatoms using a smooth Gaussian function, whereas Grim utilizes a standard clique detection algorithm to match interaction pattern graphs. Both tools are complementary and enable protein-ligand complex alignments capitalizing on both global and local pattern similarities. The new fingerprint and companion alignment tools have been successfully used in three scenarios: (i) interaction-biased alignment of protein-ligand complexes, (ii) postprocessing docking poses according to known interaction patterns for a particular target, and (iii) virtual screening for bioisosteric

  20. Photolithography-free laser-patterned HF acid-resistant chromium-polyimide mask for rapid fabrication of microfluidic systems in glass

    NASA Astrophysics Data System (ADS)

    Zamuruyev, Konstantin O.; Zrodnikov, Yuriy; Davis, Cristina E.

    2017-01-01

    Excellent chemical and physical properties of glass, over a range of operating conditions, make it a preferred material for chemical detection systems in analytical chemistry, biology, and the environmental sciences. However, it is often compromised with SU8, PDMS, or Parylene materials due to the sophisticated mask preparation requirements for wet etching of glass. Here, we report our efforts toward developing a photolithography-free laser-patterned hydrofluoric acid-resistant chromium-polyimide tape mask for rapid prototyping of microfluidic systems in glass. The patterns are defined in masking layer with a diode-pumped solid-state laser. Minimum feature size is limited to the diameter of the laser beam, 30 µm minimum spacing between features is limited by the thermal shrinkage and adhesive contact of the polyimide tape to 40 µm. The patterned glass substrates are etched in 49% hydrofluoric acid at ambient temperature with soft agitation (in time increments, up to 60 min duration). In spite of the simplicity, our method demonstrates comparable results to the other current more sophisticated masking methods in terms of the etched depth (up to 300 µm in borosilicate glass), feature under etch ratio in isotropic etch (~1.36), and low mask hole density. The method demonstrates high yield and reliability. To our knowledge, this method is the first proposed technique for rapid prototyping of microfluidic systems in glass with such high performance parameters. The proposed method of fabrication can potentially be implemented in research institutions without access to a standard clean-room facility.

  1. Directed self-assembly of proteins into discrete radial patterns

    PubMed Central

    Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

    2013-01-01

    Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

  2. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    PubMed

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions.

  3. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse.

    PubMed

    Carlson, Andreas; Mahadevan, L

    2015-12-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise.

  4. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse

    PubMed Central

    Carlson, Andreas; Mahadevan, L.

    2015-01-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise. PMID:26699430

  5. Study of local intracellular signals regulating axonal morphogenesis using a microfluidic device

    PubMed Central

    Uryu, Daiki; Tamaru, Tomohiro; Suzuki, Azusa; Sakai, Rie; Konishi, Yoshiyuki

    2016-01-01

    Abstract The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca2+ signals in axons. Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques. PMID:27877916

  6. Direct-writing colloidal photonic crystal microfluidic chips by inkjet printing for label-free protein detection.

    PubMed

    Shen, Weizhi; Li, Mingzhu; Ye, Changqing; Jiang, Lei; Song, Yanlin

    2012-09-07

    Integrating photonic crystals (PC) into microfluidic systems has attracted immense interest for its novel functions. However, it is still a great challenge to fabricate PC microfluidic chips rapidly with complex functions. In this work, a direct-writing colloidal PC microchannel was firstly achieved by inkjet printing and was used for the surface-tension-confined microfluidic immune assay. PC channels with different structure colors have been successfully integrated on one chip. The fabricated chip has the advantages of rapid fabrication, quick fluidic transport and can monitor the fluidic fluxion using the naked eye. Utilizing this PC microfluidic chip, a colorimetric label-free immune assay was realized without nonspecific adsorption interference of the target.

  7. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%.

  8. Patterns in protein primary sequences: classification, display and analysis.

    PubMed Central

    Saurugger, P. N.; Metfessel, B. A.

    1991-01-01

    The protein folding code, which is contained in the amino acid chain of a protein, has so far eluded elucidation. However, patterns of hydrophobic residues have previously been identified which show a specificity towards certain secondary structural elements. We are developing an analysis toolkit to find, visualize, and analyze patterns in primary sequences. Preliminary results show that there exist patterns in primary sequences which are useful for predicting the structural class of amino acid chains, performing especially well for the all-alpha helix and all-beta sheet classes. PMID:1807631

  9. Selective filling for patterning in microfluidic channels and integration of chromatography in "lab-on-a-chip" devices using sol-gel technology

    NASA Astrophysics Data System (ADS)

    Jindal, Rohit

    The last decade has seen tremendous advancement in the development of miniaturized chemical analysis system also known as "lab-on-a-chip". It is believed that the true potential of these devices will be achieved by integrating various functions such as separation, reaction, sensing, mixing, pumping, injection and detection onto a single chip. The ability to pattern different functionalities is indispensable for the development of highly integrated devices. In this work, a simple method based on the concept of selective filling is described for patterning in the microfluidic channels. It is based on the difference in the free energy of filling between an open and a covered part of the channel. This method was used for the integration of chromatography in the microfluidic devices. A chromatographic column was realized by utilizing sol-gel as an immobilization matrix for entrapping reversed phase chromatographic particles. Localization of the stationary phase was achieved using the selective filling technique. Channels were fabricated in quartz using photolithography and wet etching. Electroosmotic flow was used for manipulating fluid movement in the channels. Cross channel design was used for making a pulse injection of the solutes in the separation channel. An optical fiber setup was developed for carrying out on-chip UV absorbance detection. Stationary phase was created under different sol-gel synthesis conditions. It was established that the sol-gel synthesis carried out under acidic conditions provides the optimum synthesis conditions for creating separation column. Chromatographic performance of the stationary phase material was demonstrated by separating peptides present in a mixture. The sol-gel immobilization method was extended for the integration of micropump in the chip. The micropump enables pumping of the fluid in field free channels. Preliminary results, demonstrating the potential of carbon nanotubes as a support material in the microfluidic channels

  10. Predicting protein function by frequent functional association pattern mining in protein interaction networks.

    PubMed

    Cho, Young-Rae; Zhang, Aidong

    2010-01-01

    Predicting protein function from protein interaction networks has been challenging because of the complexity of functional relationships among proteins. Most previous function prediction methods depend on the neighborhood of or the connected paths to known proteins. However, their accuracy has been limited due to the functional inconsistency of interacting proteins. In this paper, we propose a novel approach for function prediction by identifying frequent patterns of functional associations in a protein interaction network. A set of functions that a protein performs is assigned into the corresponding node as a label. A functional association pattern is then represented as a labeled subgraph. Our frequent labeled subgraph mining algorithm efficiently searches the functional association patterns that occur frequently in the network. It iteratively increases the size of frequent patterns by one node at a time by selective joining, and simplifies the network by a priori pruning. Using the yeast protein interaction network, our algorithm found more than 1400 frequent functional association patterns. The function prediction is performed by matching the subgraph, including the unknown protein, with the frequent patterns analogous to it. By leave-one-out cross validation, we show that our approach has better performance than previous link-based methods in terms of prediction accuracy. The frequent functional association patterns generated in this study might become the foundations of advanced analysis for functional behaviors of proteins in a system level.

  11. Biomimetic Replication of Microscopic Metal-Organic Framework Patterns Using Printed Protein Patterns.

    PubMed

    Liang, Kang; Carbonell, Carlos; Styles, Mark J; Ricco, Raffaele; Cui, Jiwei; Richardson, Joseph J; Maspoch, Daniel; Caruso, Frank; Falcaro, Paolo

    2015-12-02

    It is demonstrated that metal-organic frameworks (MOFs) can be replicated in a biomimetic fashion from protein patterns. Bendable, fluorescent MOF patterns are formed with micrometer resolution under ambient conditions. Furthermore, this technique is used to grow MOF patterns from fingerprint residue in 30 s with high fidelity. This technique is not only relevant for crime-scene investigation, but also for biomedical applications.

  12. Microtubule patterning in the presence of moving motor proteins.

    PubMed

    White, D; de Vries, G; Martin, J; Dawes, A

    2015-10-07

    Cytoskeletal polymers such as microtubules (MTs) interact with motor proteins to form higher-order structures. In vitro experiments have shown that MT patterns such as asters, bundles, and vortices can form under the influence of a single type of dynamic motor protein. MTs also can form anti-parallel bundles, similar to bundles that form the mitotic spindle during cell division, under the influence of two types of moving motors with opposite directionality. Despite the importance of MT structures, their mechanism of formation is not yet understood. We develop an integro-partial differential equation model to describe the dynamic interactions between MTs and moving motor proteins. Our model takes into account motor protein speed, processivity, density, and directionality, as well as MT treadmilling and reorganization due to interactions with motors. Simulation results show that plus-end directed motor proteins can form vortex patterns at low motor density, while minus-end directed motor proteins form aster patterns at similar densities. Also, motor proteins with opposite directionality are able to organize MTs into anti-parallel bundles. Our model is able to provide a quantitative and qualitative description of MT patterning, providing insights into possible mechanisms of spindle formation.

  13. Excimer laser ablation for spatially controlled protein patterns

    NASA Astrophysics Data System (ADS)

    Thissen, Helmut; Hayes, Jason P.; Kingshott, Peter; Johnson, Graham; Harvey, Erol C.; Griesser, Hans J.

    2001-11-01

    Two-dimensional control over the location of proteins on surfaces is desired for a number of applications including diagnostic tests and tissue engineered medical devices. Many of these applications require patterns of specific proteins that allow subsequent two-dimensionally controlled cell attachment. The ideal technique would allow the deposition of specific protein patterns in areas where cell attachment is required, with complete prevention of unspecific protein adsorption in areas where cells are not supposed to attach. In our study, collagen I was used as an example for an extracellular matrix protein known to support the attachment of bovine corneal epithelial cells. An allylamine plasma polymer was deposited on a silicon wafer substrate, followed by grafting of poly(ethylene oxide). Two-dimensional control over the surface chemistry was achieved using a 248 nm excimer laser. Results obtained by XPS and AFM show that the combination of extremely low-fouling surfaces with excimer laser ablation can be used effectively for the production of spatially controlled protein patterns with a resolution of less than 1 micrometers . Furthermore, it was shown that bovine corneal epithelial cell attachment followed exactly the created protein patterns. The presented method is an effective tool for a number of in vitro and in vivo applications.

  14. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  15. Geometry sensing by self-organized protein patterns

    PubMed Central

    Schweizer, Jakob; Loose, Martin; Bonny, Mike; Kruse, Karsten; Mönch, Ingolf; Schwille, Petra

    2012-01-01

    In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction–diffusion system. PMID:22949703

  16. Control and automation of multilayered integrated microfluidic device fabrication.

    PubMed

    Kipper, Sarit; Frolov, Ludmila; Guy, Ortal; Pellach, Michal; Glick, Yair; Malichi, Asaf; Knisbacher, Binyamin A; Barbiro-Michaely, Efrat; Avrahami, Dorit; Yavets-Chen, Yehuda; Levanon, Erez Y; Gerber, Doron

    2017-01-31

    Integrated microfluidics is a sophisticated three-dimensional (multi layer) solution for high complexity serial or parallel processes. Fabrication of integrated microfluidic devices requires soft lithography and the stacking of thin-patterned PDMS layers. Precise layer alignment and bonding is crucial. There are no previously reported standards for alignment of the layers, which is mostly performed using uncontrolled processes with very low alignment success. As a result, integrated microfluidics is mostly used in academia rather than in the many potential industrial applications. We have designed and manufactured a semiautomatic Microfluidic Device Assembly System (μDAS) for full device production. μDAS comprises an electrooptic mechanical system consisting of four main parts: optical system, smart media holder (for PDMS), a micropositioning xyzθ system and a macropositioning XY mechanism. The use of the μDAS yielded valuable information regarding PDMS as the material for device fabrication, revealed previously unidentified errors, and enabled optimization of a robust fabrication process. In addition, we have demonstrated the utilization of the μDAS technology for fabrication of a complex 3 layered device with over 12 000 micromechanical valves and an array of 64 × 64 DNA spots on a glass substrate with high yield and high accuracy. We increased fabrication yield from 25% to about 85% with an average layer alignment error of just ∼4 μm. It also increased our protein expression yields from 80% to over 90%, allowing us to investigate more proteins per experiment. The μDAS has great potential to become a valuable tool for both advancing integrated microfluidics in academia and producing and applying microfluidic devices in the industry.

  17. Label-free study of the function of ion channel protein on a microfluidic optical sensor integrated with artificial cell membrane.

    PubMed

    Li, Zhen; Tang, Yanyan; Zhang, Ling; Wu, Jianmin

    2014-01-21

    A label-free optical sensor was constructed by integrating pH sensing material and supported phospholipid bilayers (SPBs) in a microfluidic chip. The pH sensing material was composed of a double layer structure consisting of chitosan hydrogel and electrochemically etched porous silicon. The pH change in the microchip could induce a reversible swelling of the chitosan hydrogel layer and consequently caused a shift in effective optical thickness (EOT) of the double layer, which could be observed by Fourier transformed reflectometric interference spectroscopy (FT-RIS). After phospholipid bilayers (PLBs) were self-assembled on the sensing layer, the EOT almost remained constant during the cycling of pH from 7.4 to 6.2, indicating the blockage of H(+) translocation by the PLBs. For studying the behavior of ion channel protein, gramicidin A, a typical ion channel protein, was inserted in the SPBs for mimicking the ion transportation function of cell membrane. Due to the H(+) transportation capability of gramicidin A, the optical response to pH change could partially recover. In the presence of Ca(2+), the pore of the ion channel protein was blocked, causing a significant decrease in the EOT response upon pH change. The bio-functionalized microfluidic sensor fabricated in this work will provide a reliable platform for studying the function of ion channel protein, which is an important class of drug targets.

  18. Optical microfluidics

    SciTech Connect

    Kotz, K.T.; Noble, K.A.; Faris, G.W.

    2004-09-27

    We present a method for the control of small droplets based on the thermal Marangoni effect using laser heating. With this approach, droplets covering five orders of magnitude in volume ({approx}1.7 {mu}L to 14 pL), immersed in decanol, were moved on an unmodified polystyrene surface, with speeds of up to 3 mm/s. When two droplets were brought into contact, they spontaneously fused and rapidly mixed in less than 33 ms. This optically addressed microfluidic approach has many advantages for microfluidic transport, including exceptional reconfigurability, low intersample contamination, large volume range, extremely simple substrates, no electrical connections, and ready scaling to large arrays.

  19. Modeling associated protein-DNA pattern discovery with unified scores.

    PubMed

    Chan, Tak-Ming; Lo, Leung-Yau; Sze-To, Ho-Yin; Leung, Kwong-Sak; Xiao, Xinshu; Wong, Man-Hon

    2013-01-01

    Understanding protein-DNA interactions, specifically transcription factor (TF) and transcription factor binding site (TFBS) bindings, is crucial in deciphering gene regulation. The recent associated TF-TFBS pattern discovery combines one-sided motif discovery on both the TF and the TFBS sides. Using sequences only, it identifies the short protein-DNA binding cores available only in high-resolution 3D structures. The discovered patterns lead to promising subtype and disease analysis applications. While the related studies use either association rule mining or existing TFBS annotations, none has proposed any formal unified (both-sided) model to prioritize the top verifiable associated patterns. We propose the unified scores and develop an effective pipeline for associated TF-TFBS pattern discovery. Our stringent instance-level evaluations show that the patterns with the top unified scores match with the binding cores in 3D structures considerably better than the previous works, where up to 90 percent of the top 20 scored patterns are verified. We also introduce extended verification from literature surveys, where the high unified scores correspond to even higher verification percentage. The top scored patterns are confirmed to match the known WRKY binding cores with no available 3D structures and agree well with the top binding affinities of in vivo experiments.

  20. Fourier Analysis of Conservation Patterns in Protein Secondary Structure.

    PubMed

    Palaniappan, Ashok; Jakobsson, Eric

    2017-01-01

    Residue conservation is a common observation in alignments of protein families, underscoring positions important in protein structure and function. Though many methods measure the level of conservation of particular residue positions, currently we do not have a way to study spatial oscillations occurring in protein conservation patterns. It is known that hydrophobicity shows spatial oscillations in proteins, which is characterized by computing the hydrophobic moment of the protein domains. Here, we advance the study of moments of conservation of protein families to know whether there might exist spatial asymmetry in the conservation patterns of regular secondary structures. Analogous to the hydrophobic moment, the conservation moment is defined as the modulus of the Fourier transform of the conservation function of an alignment of related protein, where the conservation function is the vector of conservation values at each column of the alignment. The profile of the conservation moment is useful in ascertaining any periodicity of conservation, which might correlate with the period of the secondary structure. To demonstrate the concept, conservation in the family of potassium ion channel proteins was analyzed using moments. It was shown that the pore helix of the potassium channel showed oscillations in the moment of conservation matching the period of the α-helix. This implied that one side of the pore helix was evolutionarily conserved in contrast to its opposite side. In addition, the method of conservation moments correctly identified the disposition of the voltage sensor of voltage-gated potassium channels to form a 310 helix in the membrane.

  1. Bioanalysis in structured microfluidic systems.

    PubMed

    Ros, Alexandra; Hellmich, Wibke; Regtmeier, Jan; Duong, Thanh Tu; Anselmetti, Dario

    2006-07-01

    Microfluidic and lab-on-a-chip devices have attracted widespread interest in separation sciences and bioanalysis. Recent designs in microfluidic devices extend common separation concepts by exploiting new phenomena for molecular dynamics on a length scale of 10 mum and below, giving rise to novel manipulation tools and nonintuitive phenomena for microseparations. Here, we focus on three very recent developments for bioseparations based on tailored microfluidic systems: Single cell navigation, trapping and steering with subsequent on-chip lysis, protein separation and LIF detection (Section 3.1), then we report dielectrophoretic trapping and separation of large DNA fragments in structured microfluidic devices (Section 3.2). Finally, a paradoxial migration phenomenon based on thermal fluctuations, periodically arranged microchannels and a biased alternating current electric field is presented in Section 3.3.

  2. Chemicals and heat generate different protein patterns in Acinetobacter calcoaceticus.

    PubMed

    Benndorf, D; Loffhagen, N; Babel, W

    1997-01-01

    The effect of exposing Acinetobacter calcoaceticus 69-V to DNP-stress and heat shock was examined by two-dimensional gel electrophoresis of proteins, which were detected either by autoradiography or by silver staining. Both DNP stress and heat shock led to altered patterns of protein synthesis or concentration. About 10% of the proteins which were synthesized newly or at an increased rate and about 25% of those which were found newly or with an increased concentration after DNP treatment were identified after heat shock, too.

  3. Integrated microfluidic platforms for investigating neuronal networks

    NASA Astrophysics Data System (ADS)

    Kim, Hyung Joon

    This dissertation describes the development and application of integrated microfluidics-based assay platforms to study neuronal activities in the nervous system in-vitro. The assay platforms were fabricated using soft lithography and micro/nano fabrication including microfluidics, surface patterning, and nanomaterial synthesis. The use of integrated microfluidics-based assay platform allows culturing and manipulating many types of neuronal tissues in precisely controlled microenvironment. Furthermore, they provide organized multi-cellular in-vitro model, long-term monitoring with live cell imaging, and compatibility with molecular biology techniques and electrophysiology experiment. In this dissertation, the integrated microfluidics-based assay platforms are developed for investigation of neuronal activities such as local protein synthesis, impairment of axonal transport by chemical/physical variants, growth cone path finding under chemical/physical cues, and synaptic transmission in neuronal circuit. Chapter 1 describes the motivation, objectives, and scope for developing in-vitro platform to study various neuronal activities. Chapter 2 introduces microfluidic culture platform for biochemical assay with large-scale neuronal tissues that are utilized as model system in neuroscience research. Chapter 3 focuses on the investigation of impaired axonal transport by beta-Amyloid and oxidative stress. The platform allows to control neuronal processes and to quantify mitochondrial movement in various regions of axons away from applied drugs. Chapter 4 demonstrates the development of microfluidics-based growth cone turning assay to elucidate the mechanism underlying axon guidance under soluble factors and shear flow. Using this platform, the behaviors of growth cone of mammalian neurons are verified under the gradient of inhibitory molecules and also shear flow in well-controlled manner. In Chapter 5, I combine in-vitro multicellular model with microfabricated MEA

  4. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms

    PubMed Central

    Otvos, Reka A.; Krishnamoorthy Iyer, Janaki; van Elk, René; Ulens, Chris; Niessen, Wilfried M. A.; Somsen, Govert W.; Kini, R. Manjunatha; Smit, August B.; Kool, Jeroen

    2015-01-01

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach. PMID:26114334

  5. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

    PubMed

    Otvos, Reka A; Iyer, Janaki Krishnamoorthy; van Elk, René; Ulens, Chris; Niessen, Wilfried M A; Somsen, Govert W; Kini, R Manjunatha; Smit, August B; Kool, Jeroen

    2015-06-24

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach.

  6. Highly efficient dynamic modification of plastic microfluidic devices using proteins in microchip capillary electrophoresis.

    PubMed

    Naruishi, Nahoko; Tanaka, Yoshihide; Higashi, Tetsuji; Wakida, Shin-ichi

    2006-10-20

    New dynamic coating agents were investigated for the manipulation of electroosmotic flow (EOF) in poly(methylmethacrylate) (PMMA) microchips. Blocking proteins designed for enzyme-linked immunosorbent assay (ELISA) applications (e.g. Block Ace and UltraBlock), and egg-white lysozyme were proposed in this study. The EOF could be enhanced, suppressed or its direction could be reversed, depending on the buffer pH and the charge on the proteins. The coating procedure is simple, requiring only filling of the microchannels with a coating solution, followed by a rinse with a running buffer solution prior to analysis. One major advantage of this method is that it is not necessary to add the coating agent to the running buffer solution. Block Ace and UltraBlock coatings were stable for at least five runs in a given microchannel without the need to condition the coating between runs other than replenishing the buffer solution after each run, i.e. the RSD values of EOF (n=5) were less than 4.3%, and there was no significant change in the EOF after 5 runs. The reproducibility of the coating procedures was found from the channel-to-channel RSD values of the EOF, and were less than 5.0% when using HEPES-Na buffer (pH 7.4) as the running buffer. Several examples of electrophoretic separations of amino acids and biogenic amines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) are demonstrated in this paper. The dynamic coating method has the potential for a broad range of applications in microchip capillary electrophoresis (microchip CE) separations.

  7. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed Central

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-01-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity. Images PMID:6286971

  8. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-06-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

  9. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-27

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

  10. Integration of dialysis membranes into a poly(dimethylsiloxane) microfluidic chip for isoelectric focusing of proteins using whole-channel imaging detection.

    PubMed

    Ou, Junjie; Glawdel, Tomasz; Samy, Razim; Wang, Shuwen; Liu, Zhen; Ren, Carolyn L; Pawliszyn, Janusz

    2008-10-01

    A poly(dimethylsiloxane) microfluidic chip-based cartridge is developed and reported here for protein analysis using isoelectic focusing (IEF)-whole-channel imaging detection (WCID) technology. In this design, commercial dialysis membranes are integrated to separate electrolytes and samples and to reduce undesired pressure-driven flow. Fused-silica capillaries are also incorporated in this design for sample injection and channel surface preconditioning. This structure is equivalent to that of a commercial fused-silica capillary-based cartridge for adapting to an IEF analyzer (iCE280 analyzer) to perform IEF-WCID. The successful integration of dialysis membranes into a microfluidic chip significantly improves IEF repeatability by eliminating undesired pressure-driven hydrodynamics and also makes sample injection much easier than that using the first-generation chip as reported recently. In this study, two microfluidic chips with a 100-microm-high, 100-microm-wide and a 200-microm-high, 50-microm-wide microchannel, respectively, were applied for qualitative and quantitative analysis of proteins. The mixture containing six pI markers with a pH range of 3-10 was successfully separated using IEF-WCID. The pH gradient exhibited a good linearity by plotting the pI value versus peak position, and the correlation coefficient reached 0.9994 and 0.9995 separately for the two chips. The separation of more complicated human hemoglobin control sample containing HbA, HbF, HbS, and HbC was also achieved. Additionally, for the quantitative analysis, a good linearity of IEF peak value versus myoglobin concentration in the range of 20-100 microg/mL was obtained.

  11. The Microfluidic Jukebox

    NASA Astrophysics Data System (ADS)

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-04-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications.

  12. The Microfluidic Jukebox

    PubMed Central

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-01-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications. PMID:24781785

  13. Photo-patterned free-standing hydrogel microarrays for massively parallel protein analysis

    NASA Astrophysics Data System (ADS)

    Duncombe, Todd A.; Herr, Amy E.

    2015-03-01

    Microfluidic technologies have largely been realized within enclosed microchannels. While powerful, a principle limitation of closed-channel microfluidics is the difficulty for sample extraction and downstream processing. To address this limitation and expand the utility of microfluidic analytical separation tools, we developed an openchannel hydrogel architecture for rapid protein analysis. Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail the development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. Owing to its open architecture - the platform can be easily interfaced with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing, mass spectroscopy). The fsPAG devices are directly photopatterened atop of and covalently attached to planar polymer or glass surfaces. Due to the fast < 1 hr design-prototype-test cycle - significantly faster than mold based fabrication techniques - rapid prototyping devices with fsPAG microstructures provides researchers a powerful tool for developing custom analytical assays. Leveraging the rapid prototyping benefits - we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAGE platform is uniquely well-suited for massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

  14. Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

    PubMed

    Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

    2014-03-21

    We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

  15. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey L. (Inventor)

    2017-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  16. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey (Inventor)

    2015-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  17. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey L. (Inventor)

    2016-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  18. Microfluidic waves

    PubMed Central

    Utz, Marcel; Begley, Matthew R.; Haj-Hariri, Hossein

    2012-01-01

    The propagation of pressure waves in fluidic channels with elastic covers is discussed in view of applications to flow control in microfluidic devices. A theory is presented which describes pressure waves in the fluid that are coupled to bending waves in the elastic cover. At low frequencies, the lateral bending of the cover dominates over longitudinal bending, leading to propagating, non-dispersive longitudinal pressure waves in the channel. The theory addresses effects due to both the finite viscosity and compressibility of the fluid. The coupled waves propagate without dispersion, as long as the wave length is larger than the channel width. It is shown that in channels of typical microfluidic dimensions, wave velocities in the range of a few 10 m s−1 result if the channels are covered by films of a compliant material such as PDMS. The application of this principle to design microfluidic band pass filters based on standing waves is discussed. Characteristic frequencies in the range of a few kHz are readily achieved with quality factors above 30. PMID:21966667

  19. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    PubMed

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  20. Microfluidic Mixing Technology for a Universal Health Sensor

    NASA Technical Reports Server (NTRS)

    Chan, Eugene Y.; Bae, Candice

    2009-01-01

    A highly efficient means of microfluidic mixing has been created for use with the rHEALTH sensor an elliptical mixer and passive curvilinear mixing patterns. The rHEALTH sensor provides rapid, handheld, complete blood count, cell differential counts, electrolyte measurements, and other lab tests based on a reusable, flow-based microfluidic platform. These geometries allow for cleaning in a reusable manner, and also allow for complete mixing of fluid streams. The microfluidic mixing is performed by flowing two streams of fluid into an elliptical or curvilinear design that allows the combination of the flows into one channel. The mixing is accomplished by either chaotic advection around micro - fluidic loops. All components of the microfluidic chip are flow-through, meaning that cleaning solution can be introduced into the chip to flush out cells, plasma proteins, and dye. Tests were performed on multiple chip geometries to show that cleaning is efficient in any flowthrough design. The conclusion from these experiments is that the chip can indeed be flushed out with microliter volumes of solution and biological samples are cleaned readily from the chip with minimal effort. The technology can be applied in real-time health monitoring at patient s bedside or in a doctor s office, and real-time clinical intervention in acute situations. It also can be used for daily measurement of hematocrit for patients on anticoagulant drugs, or to detect acute myocardial damage outside a hospital.

  1. Preparation and characterization of a packed bead immobilized trypsin reactor integrated into a PDMS microfluidic chip for rapid protein digestion.

    PubMed

    Kecskemeti, Adam; Gaspar, Attila

    2017-05-01

    This paper demonstrates the design, efficiency and applicability of a simple, inexpensive and high sample throughput microchip immobilized enzymatic reactor (IMER) for rapid protein digestion. The IMER contains conventional silica particles with covalently immobilized trypsin packed inside of a poly(dimethylsiloxane) (PDMS) microchip channel (10mm×1mm×35µm). The microchip consists of 9 different channels, enabling 9 simultaneous protein digestions. Trypsin was covalently immobilized using carbodiimide activation, the ideal trypsin/silica particle ratio (i.e. measured mass ratio before the immobilization reaction) was determined. The amount of immobilized trypsin was 10-15μg trypsin for 1mg silica particle. Migration times of CZE peptide maps showed good repeatability and reproducibility (RSD%=0.02-0.31%). The IMER maintained its activity for 2 months, in this period it was used effectively for rapid proteolysis. Four proteins (myoglobin, lysozyme, hemoglobin and albumin) in a wide size range (15-70kDa) were digested to demonstrate the applicability of the reactor. Their CZE peptide maps were compared to peptide maps obtained from standard in-solution digestion of the four proteins. The number of peptide peaks correlated well with the theoretically expected peptide number in both cases, the peak patterns of the electropherograms were similar, however, digestion with the microchip IMER requires only <10s, while in-solution digestion takes 16h. LC-MS/MS peptide mapping was also carried out, the four proteins were identified with satisfying sequence coverages (29-50%), trypsin autolysis peptides were not detected. The protein content of human serum was digested with the IMER and with in-solution digestion.

  2. Assembly of designed protein scaffolds into monolayers for nanoparticle patterning.

    PubMed

    Mejias, Sara H; Couleaud, Pierre; Casado, Santiago; Granados, Daniel; Garcia, Miguel Angel; Abad, Jose M; Cortajarena, Aitziber L

    2016-05-01

    The controlled assembly of building blocks to achieve new nanostructured materials with defined properties at different length scales through rational design is the basis and future of bottom-up nanofabrication. This work describes the assembly of the idealized protein building block, the consensus tetratricopeptide repeat (CTPR), into monolayers by oriented immobilization of the blocks. The selectivity of thiol-gold interaction for an oriented immobilization has been verified by comparing a non-thiolated protein building block. The physical properties of the CTPR protein thin biomolecular films including topography, thickness, and viscoelasticity, are characterized. Finally, the ability of these scaffolds to act as templates for inorganic nanostructures has been demonstrated by the formation of well-packed gold nanoparticles (GNPs) monolayer patterned by the CTPR monolayer.

  3. Droplet based microfluidics.

    PubMed

    Seemann, Ralf; Brinkmann, Martin; Pfohl, Thomas; Herminghaus, Stephan

    2012-01-01

    Droplet based microfluidics is a rapidly growing interdisciplinary field of research combining soft matter physics, biochemistry and microsystems engineering. Its applications range from fast analytical systems or the synthesis of advanced materials to protein crystallization and biological assays for living cells. Precise control of droplet volumes and reliable manipulation of individual droplets such as coalescence, mixing of their contents, and sorting in combination with fast analysis tools allow us to perform chemical reactions inside the droplets under defined conditions. In this paper, we will review available drop generation and manipulation techniques. The main focus of this review is not to be comprehensive and explain all techniques in great detail but to identify and shed light on similarities and underlying physical principles. Since geometry and wetting properties of the microfluidic channels are crucial factors for droplet generation, we also briefly describe typical device fabrication methods in droplet based microfluidics. Examples of applications and reaction schemes which rely on the discussed manipulation techniques are also presented, such as the fabrication of special materials and biophysical experiments.

  4. New host factors important for respiratory syncytial virus (RSV) replication revealed by a novel microfluidics screen for interactors of matrix (M) protein.

    PubMed

    Kipper, Sarit; Hamad, Samar; Caly, Leon; Avrahami, Dorit; Bacharach, Eran; Jans, David A; Gerber, Doron; Bajorek, Monika

    2015-03-01

    Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.

  5. Mapping out Min protein patterns in fully confined fluidic chambers

    PubMed Central

    Caspi, Yaron; Dekker, Cees

    2016-01-01

    The bacterial Min protein system provides a major model system for studying reaction-diffusion processes in biology. Here we present the first in vitro study of the Min system in fully confined three-dimensional chambers that are lithography-defined, lipid-bilayer coated and isolated through pressure valves. We identify three typical dynamical behaviors that occur dependent on the geometrical chamber parameters: pole-to-pole oscillations, spiral rotations, and traveling waves. We establish the geometrical selection rules and show that, surprisingly, Min-protein spiral rotations govern the larger part of the geometrical phase diagram. Confinement as well as an elevated temperature reduce the characteristic wavelength of the Min patterns, although even for confined chambers with a bacterial-level viscosity, the patterns retain a ~5 times larger wavelength than in vivo. Our results provide an essential experimental base for modeling of intracellular Min gradients in bacterial cell division as well as, more generally, for understanding pattern formation in reaction-diffusion systems. DOI: http://dx.doi.org/10.7554/eLife.19271.001 PMID:27885986

  6. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  7. Preparation of stripe-patterned heterogeneous hydrogel sheets using microfluidic devices for high-density coculture of hepatocytes and fibroblasts.

    PubMed

    Kobayashi, Aoi; Yamakoshi, Kenta; Yajima, Yuya; Utoh, Rie; Yamada, Masumi; Seki, Minoru

    2013-12-01

    Here we demonstrate the production of stripe-patterned heterogeneous hydrogel sheets for the high-density 3D coculture of multiple cell types, by using microchannel-combined micronozzle devices. The prepared hydrogel sheet, composed of multiple regions with varying physical stiffness, regulates the direction of proliferation of encapsulated cells and enables the formation of arrays of rod-like heterotypic organoids inside the hydrogel matrix. We successfully prepared stripe-patterned hydrogel sheets with a uniform thickness of ~100 μm and a width of several millimeters. Hepatoma cells (HepG2) and fibroblasts (Swiss 3T3) were embedded inside the hydrogel matrix and cocultured, to form heterotypic micro-organoids mimicking in vivo hepatic cord structures. The upregulation of hepatic functions by the 3D coculture was confirmed by analyzing liver-specific functions. The presented heterogeneous hydrogel sheet could be useful, as it provides relatively large, but precisely-controlled, 3-dimensional microenvironments for the high-density coculture of multiple types of cells.

  8. Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

    DOEpatents

    Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

    2012-12-11

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  9. Microfluidic device having an immobilized pH gradient and page gels for protein separation and analysis

    DOEpatents

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-05-20

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  10. Tacky COC: a solvent bonding technique for fabrication of microfluidic systems

    NASA Astrophysics Data System (ADS)

    Keller, Nico; Nargang, Tobias M.; Helmer, Dorothea; Rapp, Bastian E.

    2016-03-01

    The academic community knows cyclic olefin copolymer (COC) as a well suited material for microfluidic applications because COC has numerous interesting properties such as high transmittance, good chemical resistance and good biocompatibility. Here we present a fast and cost-effective method for bonding of two COC substrates: exposure to appropriate solvents gives a tacky COC surface which when brought in contact with untreated COC forms a strong and optical clear bond. The bonding process is carried out at room temperature and takes less than three minutes which makes it significantly faster than currently described methods: This method does not require special lab equipment such as hot plates or hydraulic presses. The mild conditions of the bond process also allow for such "tacky COC" lids to be used for sealing of microfluidic chips containing immobilized protein patterns which is of high interest for immunodiagnostic testing inside microfluidic chips.

  11. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  12. Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction

    PubMed Central

    Heymann, Michael; Opthalage, Achini; Wierman, Jennifer L.; Akella, Sathish; Szebenyi, Doletha M. E.; Gruner, Sol M.; Fraden, Seth

    2014-01-01

    An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation. PMID:25295176

  13. Morphology and protein patterns of honey bee drone accessory glands.

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-09-30

    We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen.

  14. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    NASA Astrophysics Data System (ADS)

    Kondrashkina, E.; Khvostichenko, D. S.; Perry, S. L.; Von Osinski, J.; Kenis, P. J. A.; Brister, K.

    2013-03-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A-1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.

  15. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    }E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.

  16. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  17. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    NASA Astrophysics Data System (ADS)

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-04-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

  18. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    DOE PAGES

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less

  19. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    SciTech Connect

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

  20. Robust regulation of oscillatory Min-protein patterns

    NASA Astrophysics Data System (ADS)

    Halatek, Jacob; Frey, Erwin

    2012-02-01

    Robust spatial patterning was crucial just from the beginning of cellular evolution, and is key to the development of multicellular organisms. In E. Coli, the oscillatory pole-to-pole dynamics of MinCDE proteins functionality prevent improper cell divisions apart from midcell. Min-oscillations are characterized by the remarkable robustness with which spatial patterns dynamically adapt to variations of cell geometry. Moreover, adaption, and therefore proper cell division, is independent of temperature. These observations raise fundamental questions about the underlying core mechanisms, and about the role of spatial cues. With a conceptually novel and universal approach to cellular geometries, we introduce a robust model based on experimental data, consistently explaining the mechanisms underlying pole-to-pole, striped and circular patterns, as well as the observed temperature-dependence. Contrary to prior conjectures, the model predicts that MinD and cardiolipin domains are not colocalized. The key mechanisms are transient sequestration of MinE, and highly canalized transfer of MinD between polar zones. MinD channeling enhances midcell localization and facilitates stripe formation, revealing the potential optimization process from which robust Min-oscillations originally arose.

  1. Microfluidic electrochemical reactors

    DOEpatents

    Nuzzo, Ralph G [Champaign, IL; Mitrovski, Svetlana M [Urbana, IL

    2011-03-22

    A microfluidic electrochemical reactor includes an electrode and one or more microfluidic channels on the electrode, where the microfluidic channels are covered with a membrane containing a gas permeable polymer. The distance between the electrode and the membrane is less than 500 micrometers. The microfluidic electrochemical reactor can provide for increased reaction rates in electrochemical reactions using a gaseous reactant, as compared to conventional electrochemical cells. Microfluidic electrochemical reactors can be incorporated into devices for applications such as fuel cells, electrochemical analysis, microfluidic actuation, pH gradient formation.

  2. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  3. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices, and which incorporates a molded ring or seal set into a ferrule cartridge, with or without a compression screw. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  4. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    the E1^E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4.

  5. Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

    PubMed

    Lin, Yen-Heng; Peng, Po-Yu

    2015-04-15

    Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.

  6. Fabrication of universal serial bus flash disk type microfluidic chip electrophoresis and application for protein analysis under ultra low voltage.

    PubMed

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei; Yuan, Hua

    2016-03-01

    A simple and effective universal serial bus (USB) flash disk type microfluidic chip electrophoresis (MCE) was developed by using poly(dimethylsiloxane) based soft lithography and dry film based printed circuit board etching techniques in this paper. The MCE had a microchannel diameter of 375 μm and an effective length of 25 mm. Equipped with a conventional online electrochemical detector, the device enabled effectively separation of bovine serum albumin, lysozyme, and cytochrome c in 80 s under the ultra low voltage from a computer USB interface. Compared with traditional capillary electrophoresis, the USB flash disk type MCE is not only portable and inexpensive but also fast with high separation efficiency.

  7. Fabrication of universal serial bus flash disk type microfluidic chip electrophoresis and application for protein analysis under ultra low voltage

    PubMed Central

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei

    2016-01-01

    A simple and effective universal serial bus (USB) flash disk type microfluidic chip electrophoresis (MCE) was developed by using poly(dimethylsiloxane) based soft lithography and dry film based printed circuit board etching techniques in this paper. The MCE had a microchannel diameter of 375 μm and an effective length of 25 mm. Equipped with a conventional online electrochemical detector, the device enabled effectively separation of bovine serum albumin, lysozyme, and cytochrome c in 80 s under the ultra low voltage from a computer USB interface. Compared with traditional capillary electrophoresis, the USB flash disk type MCE is not only portable and inexpensive but also fast with high separation efficiency. PMID:27042249

  8. Inertial microfluidic physics.

    PubMed

    Amini, Hamed; Lee, Wonhee; Di Carlo, Dino

    2014-08-07

    Microfluidics has experienced massive growth in the past two decades, and especially with advances in rapid prototyping researchers have explored a multitude of channel structures, fluid and particle mixtures, and integration with electrical and optical systems towards solving problems in healthcare, biological and chemical analysis, materials synthesis, and other emerging areas that can benefit from the scale, automation, or the unique physics of these systems. Inertial microfluidics, which relies on the unconventional use of fluid inertia in microfluidic platforms, is one of the emerging fields that make use of unique physical phenomena that are accessible in microscale patterned channels. Channel shapes that focus, concentrate, order, separate, transfer, and mix particles and fluids have been demonstrated, however physical underpinnings guiding these channel designs have been limited and much of the development has been based on experimentally-derived intuition. Here we aim to provide a deeper understanding of mechanisms and underlying physics in these systems which can lead to more effective and reliable designs with less iteration. To place the inertial effects into context we also discuss related fluid-induced forces present in particulate flows including forces due to non-Newtonian fluids, particle asymmetry, and particle deformability. We then highlight the inverse situation and describe the effect of the suspended particles acting on the fluid in a channel flow. Finally, we discuss the importance of structured channels, i.e. channels with boundary conditions that vary in the streamwise direction, and their potential as a means to achieve unprecedented three-dimensional control over fluid and particles in microchannels. Ultimately, we hope that an improved fundamental and quantitative understanding of inertial fluid dynamic effects can lead to unprecedented capabilities to program fluid and particle flow towards automation of biomedicine, materials

  9. Laterally Mobile, Functionalized Self-Assembled Monolayers at the Fluorous−Aqueous Interface in a Plug-Based Microfluidic System: Characterization and Testing with Membrane Protein Crystallization

    SciTech Connect

    Kreutz, Jason E.; Li, Liang; Roach, L. Spencer; Hatakeyama, Takuji; Ismagilov, Rustem F.

    2009-11-04

    This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous-aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.

  10. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  11. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  12. Hierarchical protein patterning by meso to molecular scale self-assembly

    NASA Astrophysics Data System (ADS)

    Andersen, Andreas S.; Sutherland, Duncan S.; Ogaki, Ryosuke

    2015-10-01

    Numerous protein patterning methodologies are used extensively for biomedical research and development. We have developed a novel bottom-up protein patterning method using a combination of self-assembly processes in the meso to molecular scale range to allow hierarchical protein patterns to be straightforwardly fabricated with low cost over large areas. As a proof of principle, we patterned vitronectin in various dimensional hierarchies using meso to nanoscale colloids and self-assembled monolayers.

  13. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  14. Superhydrophobicity for antifouling microfluidic surfaces.

    PubMed

    Shirtcliffe, N J; Roach, P

    2013-01-01

    Fouling of surfaces is often problematic in microfluidic devices, particularly when using protein or -enzymatic solutions. Various coating methods have been investigated to reduce the tendency for protein molecules to adsorb, mostly relying on hydrophobic surface chemistry or the antifouling ability of -polyethylene glycol. Here we present the potential use of superhydrophobic surfaces to not only reduce the amount of surface contamination but also to induce self-cleaning under flow conditions. The methodology is presented in order to prepare superhydrophobic surface coatings having micro- and nanoscale feature dimensions, as well as a step-by-step guide to quantify adsorbed protein down to nanogram levels. The fabrication of these surfaces as coatings via silica sol-gel and copper nano-hair growth is presented, which can be applied within microfluidic devices manufactured from various materials.

  15. PREFACE: Nano- and microfluidics Nano- and microfluidics

    NASA Astrophysics Data System (ADS)

    Jacobs, Karin

    2011-05-01

    The field of nano- and microfluidics emerged at the end of the 1990s parallel to the demand for smaller and smaller containers and channels for chemical, biochemical and medical applications such as blood and DNS analysis [1], gene sequencing or proteomics [2, 3]. Since then, new journals and conferences have been launched and meanwhile, about two decades later, a variety of microfluidic applications are on the market. Briefly, 'the small flow becomes mainstream' [4]. Nevertheless, research in nano- and microfluidics is more than downsizing the spatial dimensions. For liquids on the nanoscale, surface and interface phenomena grow in importance and may even dominate the behavior in some systems. The studies collected in this special issue all concentrate on these type of systems and were part ot the priority programme SPP1164 'Nano- and Microfluidics' of the German Science Foundation (Deutsche Forschungsgemeinschaft, DFG). The priority programme was initiated in 2002 by Hendrik Kuhlmann and myself and was launched in 2004. Friction between a moving liquid and a solid wall may, for instance, play an important role so that the usual assumption of a no-slip boundary condition is no longer valid. Likewise, the dynamic deformations of soft objects like polymers, vesicles or capsules in flow arise from the subtle interplay between the (visco-)elasticity of the object and the viscous stresses in the surrounding fluid and, potentially, the presence of structures confining the flow like channels. Consequently, new theories were developed ( see articles in this issue by Münch and Wagner, Falk and Mecke, Bonthuis et al, Finken et al, Almenar and Rauscher, Straube) and experiments were set up to unambiguously demonstrate deviations from bulk, or 'macro', behavior (see articles in this issue by Wolff et al, Vinogradova and Belyaev, Hahn et al, Seemann et al, Grüner and Huber, Müller-Buschbaum et al, Gutsche et al, Braunmüller et al, Laube et al, Brücker, Nottebrock et al

  16. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  17. Microfluidic sieve valves

    DOEpatents

    Quake, Stephen R; Marcus, Joshua S; Hansen, Carl L

    2015-01-13

    Sieve valves for use in microfluidic device are provided. The valves are useful for impeding the flow of particles, such as chromatography beads or cells, in a microfluidic channel while allowing liquid solution to pass through the valve. The valves find particular use in making microfluidic chromatography modules.

  18. Tunable Microfluidic Microlasers

    DTIC Science & Technology

    2011-09-01

    particularly convenient material for microfluidic experiments with LC. Figure 7: A droplet of E7 nematic liquid crystal on a PDMS...AFRL-AFOSR-UK-TR-2011-0039 TUNABLE MICROFLUIDIC MICROLASERS Francesco Simoni Universita Politecnica delle Marche...DATES COVERED (From – To) 15 June 2010 – 15 June 2011 4. TITLE AND SUBTITLE TUNABLE MICROFLUIDIC MICROLASERS 5a. CONTRACT NUMBER FA8655

  19. Microfluidic System for Solution Array Based Bioassays

    SciTech Connect

    Dougherty, G M; Tok, J B; Pannu, S S; Rose, K A

    2006-02-10

    The objective of this project is to demonstrate new enabling technology for multiplex biodetection systems that are flexible, miniaturizable, highly automated, low cost, and high performance. It builds on prior successes at LLNL with particle-based solution arrays, such as those used in the Autonomous Pathogen Detection System (APDS) successfully field deployed to multiple locations nationwide. We report the development of a multiplex solution array immunoassay based upon engineered metallic nanorod particles. Nanobarcodes{reg_sign} particles are fabricated by sequential electrodeposition of dissimilar metals within porous alumina templates, yielding optically encoded striping patterns that can be read using standard laboratory microscope optics and PC-based image processing software. The addition of self-assembled monolayer (SAM) coatings and target-specific antibodies allows each encoded class of nanorod particles to be directed against a different antigen target. A prototype assay panel directed against bacterial, viral, and soluble protein targets demonstrates simultaneous detection at sensitivities comparable to state of the art immunoassays, with minimal cross-reactivity. Studies have been performed to characterize the colloidal properties (zeta potential) of the suspended nanorod particles as a function of pH, the ionic strength of the suspending solution, and surface functionalization state. Additional studies have produced means for the non-contact manipulation of the particles, including the insertion of magnetic nickel stripes within the encoding pattern, and control via externally applied electromagnetic fields. Using the results of these studies, the novel Nanobarcodes{reg_sign} based assay was implemented in a prototype automated system with the sample processing functions and optical readout performed on a microfluidic card. The unique physical properties of the nanorod particles enable the development of integrated microfluidic systems for

  20. Electro-Microfluidic Packaging

    NASA Astrophysics Data System (ADS)

    Benavides, G. L.; Galambos, P. C.

    2002-06-01

    There are many examples of electro-microfluidic products that require cost effective packaging solutions. Industry has responded to a demand for products such as drop ejectors, chemical sensors, and biological sensors. Drop ejectors have consumer applications such as ink jet printing and scientific applications such as patterning self-assembled monolayers or ejecting picoliters of expensive analytes/reagents for chemical analysis. Drop ejectors can be used to perform chemical analysis, combinatorial chemistry, drug manufacture, drug discovery, drug delivery, and DNA sequencing. Chemical and biological micro-sensors can sniff the ambient environment for traces of dangerous materials such as explosives, toxins, or pathogens. Other biological sensors can be used to improve world health by providing timely diagnostics and applying corrective measures to the human body. Electro-microfluidic packaging can easily represent over fifty percent of the product cost and, as with Integrated Circuits (IC), the industry should evolve to standard packaging solutions. Standard packaging schemes will minimize cost and bring products to market sooner.

  1. Fabrication of nanometer-sized protein patterns using atomic force microscopy and selective immobilization.

    PubMed Central

    Wadu-Mesthrige, K; Amro, N A; Garno, J C; Xu, S; Liu , G

    2001-01-01

    A new methodology is introduced to produce nanometer-sized protein patterns. The approach includes two main steps, nanopatterning of self-assembled monolayers using atomic force microscopy (AFM)-based nanolithography and subsequent selective immobilization of proteins on the patterned monolayers. The resulting templates and protein patterns are characterized in situ using AFM. Compared with conventional protein fabrication methods, this approach is able to produce smaller patterns with higher spatial precision. In addition, fabrication and characterization are completed in near physiological conditions. The adsorption configuration and bioreactivity of the proteins within the nanopatterns are also studied in situ. PMID:11259301

  2. Design of hydrodynamically confined microfluidics: controlling flow envelope and pressure.

    PubMed

    Christ, Kevin V; Turner, Kevin T

    2011-04-21

    Closed-channel microfluidic devices are widely used in a number of chemical and biological applications; however, it is often difficult to interact with samples, such as cells, that are enclosed inside them. Hydrodynamically confined microflows (HCMs) allow microfluidic-type flows to be generated in open liquid environments, such as Petri dishes, thus greatly increasing the flexibility of microfluidic approaches. HCMs have previously been used for protein patterning and selective cell treatment applications, but the underlying fluid mechanics is not fully understood. Here, we examine the effect of device geometry and flow parameters on the properties of the flow envelope and pressure drop of several two-port HCM devices using a combination of experiments and modeling. A three-port device, which allows for different flow envelope shapes to be generated, is also analyzed. The experimental results agree well with the 3-D computational fluid dynamics simulations, with the majority of the measurements within 10% of the simulations. The results presented provide a framework for understanding the fluid mechanics of HCMs and will aid in the design of HCM devices for a broad range of applications.

  3. Protein patterning on silicon-based surface using background hydrophobic thin film.

    PubMed

    Lee, Chang-Soo; Lee, Sang-Ho; Park, Sung-Soo; Kim, Yong-Kweon; Kim, Byung-Gee

    2003-04-01

    A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.

  4. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    PubMed Central

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-01-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting. PMID:26439164

  5. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    NASA Astrophysics Data System (ADS)

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-10-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting.

  6. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  7. Infrared light induced patterning of proteins on ppNIPAM thermoresponsive thin films: a "protein laser printer".

    PubMed

    Cheng, Xuanhong; Yegan Erdem, E; Takeuchi, Shoji; Fujita, Hiroyuki; Ratner, Buddy D; Böhringer, Karl F

    2010-04-21

    Protein micropatterns have applications in fundamental life sciences and clinical medicine. In this work, we present a new technique to create 2-D protein micropatterns by local activation of a thin film of thermoresponsive plasma-deposited poly(N-isopropylacrylamide) (ppNIPAM) using a computer-controlled infrared laser beam. While the whole substrate is exposed to the protein solution, protein deposition happens only at laser-activated locations. A few seconds of laser exposure is all that is required to form a pattern with resolution in the single micrometre range. Successful ligand binding after protein deposition indicates that protein function remains intact after laser-induced adsorption onto ppNIPAM. This rapid, simple technique advances currently available strategies for protein patterning by its potential to pattern proteins in an enclosed environment or onto a 3-D scaffold.

  8. Hierarchical polymer brush nanoarrays: a versatile way to prepare multiscale patterns of proteins.

    PubMed

    Li, Yunfeng; Zhang, Junhu; Liu, Wendong; Li, Daowei; Fang, Liping; Sun, Hongchen; Yang, Bai

    2013-03-01

    This paper presents a versatile way to prepare multiscale and gradient patterns of proteins. The protein patterns are fabricated by conjugating proteins covalently on patterns of polymer brush that are prepared by techniques combining colloidal lithography with photolithography, and two-step colloidal lithography. Taking advantages of this technique, the parameters of protein patterns, such as height, diameters, periods, and distances between two dots, can be arbitrarily tuned. In addition, the protein patterns with varies of architectures, such as microdiscs, microstripes, microrings, microtriangles, microgrids, etc., consisting of protein nanodots, are prepared and the sample size is up to 4 cm(2). The as-prepared patterns of fibronectin can promote the cell adhesion and cell location.

  9. Ultrafast microfluidics using surface acoustic waves

    PubMed Central

    Yeo, Leslie Y.; Friend, James R.

    2009-01-01

    We demonstrate that surface acoustic waves (SAWs), nanometer amplitude Rayleigh waves driven at megahertz order frequencies propagating on the surface of a piezoelectric substrate, offer a powerful method for driving a host of extremely fast microfluidic actuation and micro∕bioparticle manipulation schemes. We show that sessile drops can be translated rapidly on planar substrates or fluid can be pumped through microchannels at 1–10 cm∕s velocities, which are typically one to two orders quicker than that afforded by current microfluidic technologies. Through symmetry-breaking, azimuthal recirculation can be induced within the drop to drive strong inertial microcentrifugation for micromixing and particle concentration or separation. Similar micromixing strategies can be induced in the same microchannel in which fluid is pumped with the SAW by merely changing the SAW frequency to rapidly switch the uniform through-flow into a chaotic oscillatory flow by exploiting superpositioning of the irradiated sound waves from the sidewalls of the microchannel. If the flow is sufficiently quiescent, the nodes of the transverse standing wave that arises across the microchannel also allow for particle aggregation, and hence, sorting on nodal lines. In addition, the SAW also facilitates other microfluidic capabilities. For example, capillary waves excited at the free surface of a sessile drop by the SAW underneath it can be exploited for micro∕nanoparticle collection and sorting at nodal points or lines at low powers. At higher powers, the large accelerations off the substrate surface as the SAW propagates across drives rapid destabilization of the drop free surface giving rise to inertial liquid jets that persist over 1–2 cm in length or atomization of the entire drop to produce 1–10 μm monodispersed aerosol droplets, which can be exploited for ink-jet printing, mass spectrometry interfacing, or pulmonary drug delivery. The atomization of polymer∕protein solutions

  10. Manufacturing methods and applications of membranes in microfluidics.

    PubMed

    Chen, Xueye; Shen, Jienan; Hu, Zengliang; Huo, Xuyao

    2016-12-01

    Applications of membranes in microfluidics solved many thorny problems for analytical chemistry and bioscience, so that the use of membranes in microfluidics has been a topic of growing interest. Many different examples have been reported, demonstrating the versatile use of membranes. This work reviews a lot of applications of membranes in microfluidics. Membranes in microfluidics for applications including chemical reagents detection, gas detection, drug screening, cell, protein, microreactor, electrokinetical fluid, pump and valve and fluid transport control and so on, have been analyzed and discussed. In addition, the definition and basic concepts of membranes are summed up. And the methods of manufacturing membranes in microfluidics are discussed. This paper will provide a helpful reference to researchers who want to study applications of membranes in microfluidics.

  11. Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

    PubMed Central

    Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

    2015-01-01

    In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

  12. Digital Microfluidics Sample Analyzer

    NASA Technical Reports Server (NTRS)

    Pollack, Michael G.; Srinivasan, Vijay; Eckhardt, Allen; Paik, Philip Y.; Sudarsan, Arjun; Shenderov, Alex; Hua, Zhishan; Pamula, Vamsee K.

    2010-01-01

    Three innovations address the needs of the medical world with regard to microfluidic manipulation and testing of physiological samples in ways that can benefit point-of-care needs for patients such as premature infants, for which drawing of blood for continuous tests can be life-threatening in their own right, and for expedited results. A chip with sample injection elements, reservoirs (and waste), droplet formation structures, fluidic pathways, mixing areas, and optical detection sites, was fabricated to test the various components of the microfluidic platform, both individually and in integrated fashion. The droplet control system permits a user to control droplet microactuator system functions, such as droplet operations and detector operations. Also, the programming system allows a user to develop software routines for controlling droplet microactuator system functions, such as droplet operations and detector operations. A chip is incorporated into the system with a controller, a detector, input and output devices, and software. A novel filler fluid formulation is used for the transport of droplets with high protein concentrations. Novel assemblies for detection of photons from an on-chip droplet are present, as well as novel systems for conducting various assays, such as immunoassays and PCR (polymerase chain reaction). The lab-on-a-chip (a.k.a., lab-on-a-printed-circuit board) processes physiological samples and comprises a system for automated, multi-analyte measurements using sub-microliter samples of human serum. The invention also relates to a diagnostic chip and system including the chip that performs many of the routine operations of a central labbased chemistry analyzer, integrating, for example, colorimetric assays (e.g., for proteins), chemiluminescence/fluorescence assays (e.g., for enzymes, electrolytes, and gases), and/or conductometric assays (e.g., for hematocrit on plasma and whole blood) on a single chip platform.

  13. A microfluidic platform with a flow-balanced fluidic network for osteoarthritis diagnosis

    NASA Astrophysics Data System (ADS)

    Kim, Kangil; Park, Yoo Min; Yoon, Hyun C.; Yang, Sang Sik

    2013-05-01

    Osteoarthritis (OA) is one of the most common human diseases, and the occurrence of OA is likely to increase with the increase of population ages. The diagnosis of OA is based on patientrelevant measures, structural measures, and measurement of biomarkers that are released through joint metabolism. Traditionally, radiography or magnetic resonance imaging (MRI) is used to diagnose OA and predict its course. However, diagnostic imaging in OA provides only indirect information on pathology and treatment response. A sensing of OA based on the detection of biomarkers insignificantly improves the accuracy and sensitivity of diagnosis and reduces the cost compared with that of radiography or MRI. In our former study, we proposed microfluidic platform to detect biomarker of OA. But the platform can detect only one biomarker because it has one microfluidic channel. In this report, we proposes microfluidic platform that can detect several biomarkers. The proposed platform has three layers. The bottom layer has gold patterns on a Si substrate for optical sensing. The middle layer and top layer were fabricated by polydimethysiloxane (PDMS) using soft-lithography. The middle layer has four channels connecting top layer to bottom layer. The top layer consists of one sample injection inlet, and four antibody injection inlets. To this end, we designed a flow-balanced microfluidic network using analogy between electric and hydraulic systems. Also, the designed microfluidic network was confirmed by finite element model (FEM) analysis using COMSOL FEMLAB. To verify the efficiency of fabricated platform, the optical sensing test was performed to detect biomarker of OA using fluorescence microscope. We used cartilage oligomeric matrix protein (COMP) as biomarker because it reflects specific changes in joint tissues. The platform successfully detected various concentration of COMP (0, 100, 500, 1000 ng/ml) at each chamber. The effectiveness of the microfluidic platform was verified

  14. Fabrication and characterization of mesoscale protein patterns using atomic force microscopy (AFM)

    NASA Astrophysics Data System (ADS)

    Gao, Pei

    2011-07-01

    A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns. KEYWORDS: Atomic Force Microscopy (AFM), Protein Patterns, Lysozyme, Aptamer

  15. Protein kinase C modulates ventilatory patterning in the developing rat.

    PubMed

    Bandla, H P; Simakajornboon, N; Graff, G R; Gozal, D

    1999-03-01

    Protein kinase C (PKC) mediates important components of signal transduction pathways underlying neuronal excitability and modulates respiratory timing mechanisms in adult rats. To determine ventilatory effects of systemic PKC inhibition during development, whole-body plethysmographic recordings were conducted in 2-3-d (n = 11), 5-6-d (n = 19), 10-12-d (n = 14), and 20-21-d-old (n = 14) rat pups after treatment with vehicle and Ro 32-0432 (100 mg/kg, intraperitoneally). Ro 32-0432 decreased minute ventilation (V E) by 51.0 +/- 5.5% (mean +/- SEM) in youngest pups (p < 0.01) but only 19.1 +/- 6.8% in 20-21-d-old pups (p < 0.01). V E decreases were always due to frequency reductions with tidal volume (VT) remaining unaffected. Respiratory rate decreases primarily resulted from marked expiratory time (TE) prolongations being more pronounced in 2-3-d-old (115.5 +/- 28.9%) compared with 20-21-d old (36.6 +/- 10.9%; p < 0.002 analysis of variance [ANOVA] ). Expression of the PKC isoforms alpha, beta, gamma, delta, iota, and mu was further examined in brainstem and cortex by immunoblotting and revealed different patterns with postnatal age and location. We conclude that endogenous PKC inhibition elicits age-dependent ventilatory reductions which primarily affect timing mechanisms rather than changes in volume drive. This effect on ventilation abates with increasing postnatal age suggesting that the neural substrate mediating overall respiratory output may be more critically dependent on PKC activity in the immature animal.

  16. Rapid wasted-free microfluidic fabrication based on ink-jet approach for microfluidic sensing applications

    NASA Astrophysics Data System (ADS)

    Jarujareet, Ungkarn; Amarit, Rattasart; Sumriddetchkajorn, Sarun

    2016-11-01

    Realizing that current microfluidic chip fabrication techniques are time consuming and labor intensive as well as always have material leftover after chip fabrication, this research work proposes an innovative approach for rapid microfluidic chip production. The key idea relies on a combination of a widely-used inkjet printing method and a heat-based polymer curing technique with an electronic-mechanical control, thus eliminating the need of masking and molds compared to typical microfluidic fabrication processes. In addition, as the appropriate amount of polymer is utilized during printing, there is much less amount of material wasted. Our inkjet-based microfluidic printer can print out the desired microfluidic chip pattern directly onto a heated glass surface, where the printed polymer is suddenly cured. Our proof-of-concept demonstration for widely-used single-flow channel, Y-junction, and T-junction microfluidic chips shows that the whole microfluidic chip fabrication process requires only 3 steps with a fabrication time of 6 minutes.

  17. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography.

    PubMed

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y; Maynard, Heather D

    2015-03-20

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  18. Trehalose Glycopolymer Resists Allow Direct Writing of Protein Patterns by Electron-Beam Lithography

    PubMed Central

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-01-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein we present a new resist that protects proteins during electron beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively cross-link to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometer and nanometer scale without requiring cleanroom conditions. PMID:25791943

  19. Integrated Microfluidic Reactors.

    PubMed

    Lin, Wei-Yu; Wang, Yanju; Wang, Shutao; Tseng, Hsian-Rong

    2009-12-01

    Microfluidic reactors exhibit intrinsic advantages of reduced chemical consumption, safety, high surface-area-to-volume ratios, and improved control over mass and heat transfer superior to the macroscopic reaction setting. In contract to a continuous-flow microfluidic system composed of only a microchannel network, an integrated microfluidic system represents a scalable integration of a microchannel network with functional microfluidic modules, thus enabling the execution and automation of complicated chemical reactions in a single device. In this review, we summarize recent progresses on the development of integrated microfluidics-based chemical reactors for (i) parallel screening of in situ click chemistry libraries, (ii) multistep synthesis of radiolabeled imaging probes for positron emission tomography (PET), (iii) sequential preparation of individually addressable conducting polymer nanowire (CPNW), and (iv) solid-phase synthesis of DNA oligonucleotides. These proof-of-principle demonstrations validate the feasibility and set a solid foundation for exploring a broad application of the integrated microfluidic system.

  20. 3D thermoplastic elastomer microfluidic devices for biological probe immobilization.

    PubMed

    Brassard, Daniel; Clime, Liviu; Li, Kebin; Geissler, Matthias; Miville-Godin, Caroline; Roy, Emmanuel; Veres, Teodor

    2011-12-07

    Microfluidics has emerged as a valuable tool for the high-resolution patterning of biological probes on solid supports. Yet, its widespread adoption as a universal biological immobilization tool is still limited by several technical challenges, particularly for the patterning of isolated spots using three-dimensional (3D) channel networks. A key limitation arises from the difficulties to adapt the techniques and materials typically used in prototyping to low-cost mass-production. In this paper, we present the fabrication of thin thermoplastic elastomer membranes with microscopic through-holes using a hot-embossing process that is compatible with high-throughput manufacturing. The membranes provide the basis for the fabrication of highly integrated 3D microfluidic devices with a footprint of only 1 × 1 cm(2). When placed on a solid support, the device allows for the immobilization of up to 96 different probes in the form of a 10 × 10 array comprising isolated spots of 50 × 50 μm(2). The design of the channel network is optimized using 3D simulations based on the Lattice-Boltzmann method to promote capillary action as the sole force distributing the liquid in the device. Finally, we demonstrate the patterning of DNA and protein arrays on hard thermoplastic substrates yielding spots of excellent definition that prove to be highly specific in subsequent hybridization experiments.

  1. Predicting Droplet Formation on Centrifugal Microfluidic Platforms

    NASA Astrophysics Data System (ADS)

    Moebius, Jacob Alfred

    Centrifugal microfluidics is a widely known research tool for biological sample and water quality analysis. Currently, the standard equipment used for such diagnostic applications include slow, bulky machines controlled by multiple operators. These machines can be condensed into a smaller, faster benchtop sample-to-answer system. Sample processing is an important step taken to extract, isolate, and convert biological factors, such as nucleic acids or proteins, from a raw sample to an analyzable solution. Volume definition is one such step. The focus of this thesis is the development of a model predicting monodispersed droplet formation and the application of droplets as a technique for volume definition. First, a background of droplet microfluidic platforms is presented, along with current biological analysis technologies and the advantages of integrating such technologies onto microfluidic platforms. Second, background and theories of centrifugal microfluidics is given, followed by theories relevant to droplet emulsions. Third, fabrication techniques for centrifugal microfluidic designs are discussed. Finally, the development of a model for predicting droplet formation on the centrifugal microfluidic platform are presented for the rest of the thesis. Predicting droplet formation analytically based on the volumetric flow rates of the continuous and dispersed phases, the ratios of these two flow rates, and the interfacial tension between the continuous and dispersed phases presented many challenges, which will be discussed in this work. Experimental validation was completed using continuous phase solutions of different interfacial tensions. To conclude, prospective applications are discussed with expected challenges.

  2. Patterned polymer nanowire arrays as an effective protein immobilizer for biosensing and HIV detection

    NASA Astrophysics Data System (ADS)

    Shen, Yue; Liu, Yingyi; Zhu, Guang; Fang, Hao; Huang, Yunhui; Jiang, Xingyu; Wang, Zhong L.

    2012-12-01

    We report an array of polymeric nanowires for effectively immobilizing biomolecules on biochips owing to the large surface area. The nanowires were fabricated in predesigned patterns using an inductively coupled plasma (ICP) etching process. Microfluidic biochips integrated using the substrates with arrays of nanowires and polydimethylsiloxane channels have been demonstrated to be effective for detecting antigens, and a detection limit of antigens at 0.2 μg mL-1 has been achieved, which is improved by a factor of 50 compared to that based on flat substrates without the nanowires. In addition, the high sensitivity for clinical detection of human immunodeficiency virus (HIV) antibody has also been demonstrated, showing a 20 times enhancement in fluorescent signal intensity between the samples with positive and negative HIV.

  3. Microfluidics for manipulating cells.

    PubMed

    Mu, Xuan; Zheng, Wenfu; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2013-01-14

    Microfluidics, a toolbox comprising methods for precise manipulation of fluids at small length scales (micrometers to millimeters), has become useful for manipulating cells. Its uses range from dynamic management of cellular interactions to high-throughput screening of cells, and to precise analysis of chemical contents in single cells. Microfluidics demonstrates a completely new perspective and an excellent practical way to manipulate cells for solving various needs in biology and medicine. This review introduces and comments on recent achievements and challenges of using microfluidics to manipulate and analyze cells. It is believed that microfluidics will assume an even greater role in the mechanistic understanding of cell biology and, eventually, in clinical applications.

  4. Micro-fluidic interconnect

    DOEpatents

    Okandan, Murat; Galambos, Paul C.; Benavides, Gilbert L.; Hetherington, Dale L.

    2006-02-28

    An apparatus for simultaneously aligning and interconnecting microfluidic ports is presented. Such interconnections are required to utilize microfluidic devices fabricated in Micro-Electromechanical-Systems (MEMS) technologies, that have multiple fluidic access ports (e.g. 100 micron diameter) within a small footprint, (e.g. 3 mm.times.6 mm). Fanout of the small ports of a microfluidic device to a larger diameter (e.g. 500 microns) facilitates packaging and interconnection of the microfluidic device to printed wiring boards, electronics packages, fluidic manifolds etc.

  5. Easy fabrication of thin membranes with through holes. Application to protein patterning.

    PubMed

    Masters, Thomas; Engl, Wilfried; Weng, Zhe L; Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm(2). Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy.

  6. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  7. Parallel imaging microfluidic cytometer.

    PubMed

    Ehrlich, Daniel J; McKenna, Brian K; Evans, James G; Belkina, Anna C; Denis, Gerald V; Sherr, David H; Cheung, Man Ching

    2011-01-01

    By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of fluorescence-activated flow cytometry (FCM) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity, and (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery. The PMC promises to relieve a bottleneck in these previously constrained applications. The PMC may also be a powerful tool for finding rare primary cells in the clinic. The multichannel architecture of current PMC prototypes allows 384 unique samples for a cell-based screen to be read out in ∼6-10 min, about 30 times the speed of most current FCM systems. In 1D intracellular imaging, the PMC can obtain protein localization using HCS marker strategies at many times for the sample throughput of charge-coupled device (CCD)-based microscopes or CCD-based single-channel flow cytometers. The PMC also permits the signal integration time to be varied over a larger range than is practical in conventional flow cytometers. The signal-to-noise advantages are useful, for example, in counting rare positive cells in the most difficult early stages of genome-wide screening. We review the status of parallel microfluidic cytometry and discuss some of the directions the new technology may take.

  8. Microfluidic devices for cell cultivation and proliferation

    PubMed Central

    Tehranirokh, Masoomeh; Kouzani, Abbas Z.; Francis, Paul S.; Kanwar, Jagat R.

    2013-01-01

    Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined. PMID:24273628

  9. THE USE OF EVOLUTIONARY PATTERNS IN PROTEIN ANNOTATION

    PubMed Central

    Wilkins, Angela; Bachman, Ben; Erdin, Serkan; Lichtarge, Olivier

    2012-01-01

    Summary With genomic data skyrocketing, their biological interpretation remains a serious challenge. Diverse computational methods address this problem by pointing to the existence of recurrent patterns among sequence, structure, and function. These patterns emerge naturally from evolutionary variation, natural selection, and divergence—the defining features of biological systems—and they identify molecular events and shapes that underlie specificity of function and allosteric communication. Here we review these methods, and the patterns they identify in case studies and in proteome-wide applications, to infer and rationally redesign function. PMID:22633559

  10. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  11. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda.

    PubMed

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-Bin; Ye, Jin-Zhou; Yang, Man-Jun; Jiang, Ming; Peng, Xuan-Xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

  12. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  13. Unconventional microfluidics: expanding the discipline.

    PubMed

    Nawaz, Ahmad Ahsan; Mao, Xiaole; Stratton, Zackary S; Huang, Tony Jun

    2013-04-21

    Since its inception, the discipline of microfluidics has been harnessed for innovations in the biomedicine/chemistry fields-and to great effect. This success has had the natural side-effect of stereotyping microfluidics as a platform for medical diagnostics and miniaturized lab processes. But microfluidics has more to offer. And very recently, some researchers have successfully applied microfluidics to fields outside its traditional domains. In this Focus article, we highlight notable examples of such "unconventional" microfluidics applications (e.g., robotics, electronics). It is our hope that these early successes in unconventional microfluidics prompt further creativity, and inspire readers to expand the microfluidics discipline.

  14. Unconventional microfluidics: expanding the discipline

    PubMed Central

    Nawaz, Ahmad Ahsan; Mao, Xiaole; Stratton, Zackary S.; Huang, Tony Jun

    2014-01-01

    Since its inception, the discipline of microfluidics has been harnessed for innovations in the biomedicine/chemistry fields—and to great effect. This success has had the natural side-effect of stereotyping microfluidics as a platform for medical diagnostics and miniaturized lab processes. But microfluidics has more to offer. And very recently, some researchers have successfully applied microfluidics to fields outside its traditional domains. In this Focus article, we highlight notable examples of such “unconventional” microfluidics applications (e.g., robotics, electronics). It is our hope that these early successes in unconventional microfluidics prompt further creativity, and inspire readers to expand the microfluidics discipline. PMID:23478651

  15. Reagent-loaded plastic microfluidic chips for detecting homocysteine

    NASA Astrophysics Data System (ADS)

    Suk, Ji Won; Jang, Jae-Young; Cho, Jun-Hyeong

    2008-05-01

    This report describes the preliminary study on plastic microfluidic chips with pre-loaded reagents for detecting homocysteine (Hcy). All reagents needed in an Hcy immunoassay were included in a microfluidic chip to remove tedious assay steps. A simple and cost-effective bonding method was developed to realize reagent-loaded microfluidic chips. This technique uses an intermediate layer between two plastic substrates by selectively patterning polydimethylsiloxane (PDMS) on the embossed surface of microchannels and fixing the substrates under pressure. Using this bonding method, the competitive immunoassay for SAH, a converted form of Hcy, was performed without any damage to reagents in chips, and the results showed that the fluorescent signal from antibody antigen binding decreased as the SAH concentration increased. Based on the SAH immunoassay, whole immunoassay steps for Hcy detection were carried out in plastic microfluidic chips with all necessary reagents. These experiments demonstrated the feasibility of the Hcy immunoassay in microfluidic devices.

  16. Modulation of cell adhesion complexes by surface protein patterns.

    PubMed

    Pesen, Devrim; Haviland, David B

    2009-03-01

    Cell adhesion is an important process in several biological phenomena. To investigate the formation and organization of focal adhesions, we developed a patterning approach based on electron beam lithography. Nanodots (radius <1230 nm) and nanorings (inner radius <320 nm) of fibronectin (FN) were patterned on a K-Casein background. Intracellular vinculin immunofluorescence mirrored the FN nanopatterns. Atomic force microscopy showed that FN nanodots and nanorings organize the immediate cytoskeleton into straight fibrils and diverging fibril bundles, respectively. Our results suggest that a minimum of approximately 40 FN molecules is required for a cell to form a focal adhesion.

  17. Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum-Pressure Accelerated Movement (V-PAM).

    PubMed

    Yu, Zeta Tak For; Cheung, Mei Ki; Liu, Shirley Xiaosu; Fu, Jianping

    2016-09-01

    Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum-Pressure Accelerated Movement (V-PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead-end channels and closed chambers. Operation of the V-PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas-permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V-PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V-PAM for rapid filling of temperature-sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V-PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs-on-chips, and biomimetic studies.

  18. Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

    PubMed

    Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

    2016-11-16

    DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

  19. Image reversal for direct electron beam patterning of protein coated surfaces.

    PubMed

    Pesen, Devrim; Erlandsson, Anna; Ulfendahl, Mats; Haviland, David B

    2007-11-01

    Electron beam lithography (EBL) is used to create surfaces with protein patterns, which are characterized by immunofluorescence and atomic force microscopies. Both negative and positive image processes are realized by electron beam irradiation of proteins absorbed on a silicon surface, where image reversal is achieved by selectively binding a second species of protein to the electron beam exposed areas on the first protein layer. Biofunctionality at the cellular level was established by culturing cortical cells on patterned lines of fibronectin adsorbed on a bovine serum albumin background for 7 days in culture.

  20. Microfluidic Diffusion Viscometer for Rapid Analysis of Complex Solutions.

    PubMed

    Arosio, Paolo; Hu, Kevin; Aprile, Francesco A; Müller, Thomas; Knowles, Tuomas P J

    2016-04-05

    The viscosity of complex solutions is a physical property of central relevance for a large number of applications in material, biological, and biotechnological sciences. Here we demonstrate a microfluidic technology to measure the viscosity of solutions by following the advection and diffusion of tracer particles under steady-state flow. We validate our method with standard water-glycerol mixtures, and then we apply this microfluidic diffusion viscometer to measure the viscosity of protein solutions at high concentrations as well as of a crude cell lysate. Our approach exhibits a series of attractive features, including analysis time on the order of seconds and the consumption of a few μL of sample, as well as the possibility to readily integrate the microfluidic viscometer in other instrument platforms or modular microfluidic devices. These characteristics make microfluidic diffusion viscometry an attractive approach in automated processes in biotechnology and health-care sciences where fast measurements with limited amount of sample consumption are required.

  1. Microfluidics and microbial engineering.

    PubMed

    Kou, Songzi; Cheng, Danhui; Sun, Fei; Hsing, I-Ming

    2016-02-07

    The combination of microbial engineering and microfluidics is synergistic in nature. For example, microfluidics is benefiting from the outcome of microbial engineering and many reported point-of-care microfluidic devices employ engineered microbes as functional parts for the microsystems. In addition, microbial engineering is facilitated by various microfluidic techniques, due to their inherent strength in high-throughput screening and miniaturization. In this review article, we firstly examine the applications of engineered microbes for toxicity detection, biosensing, and motion generation in microfluidic platforms. Secondly, we look into how microfluidic technologies facilitate the upstream and downstream processes of microbial engineering, including DNA recombination, transformation, target microbe selection, mutant characterization, and microbial function analysis. Thirdly, we highlight an emerging concept in microbial engineering, namely, microbial consortium engineering, where the behavior of a multicultural microbial community rather than that of a single cell/species is delineated. Integrating the disciplines of microfluidics and microbial engineering opens up many new opportunities, for example in diagnostics, engineering of microbial motors, development of portable devices for genetics, high throughput characterization of genetic mutants, isolation and identification of rare/unculturable microbial species, single-cell analysis with high spatio-temporal resolution, and exploration of natural microbial communities.

  2. Applying microfluidics to electrophysiology.

    PubMed

    Eddington, David T

    2007-01-01

    Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities. Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice. By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice. This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate. This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate. These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants. This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

  3. Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

    SciTech Connect

    Valley, Cary T.; Porter, Douglas F.; Qiu, Chen; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2012-06-28

    mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

  4. Integrated multifunctional microfluidics for automated proteome analyses.

    PubMed

    Osiri, John K; Shadpour, Hamed; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    Proteomics is a challenging field for realizing totally integrated microfluidic systems for complete proteome processing due to several considerations, including the sheer number of different protein types that exist within most proteomes, the large dynamic range associated with these various protein types, and the diverse chemical nature of the proteins comprising a typical proteome. For example, the human proteome is estimated to have >10(6) different components with a dynamic range of >10(10). The typical processing pipeline for proteomics involves the following steps: (1) selection and/or extraction of the particular proteins to be analyzed; (2) multidimensional separation; (3) proteolytic digestion of the protein sample; and (4) mass spectral identification of either intact proteins (top-down proteomics) or peptide fragments generated from proteolytic digestions (bottom-up proteomics). Although a number of intriguing microfluidic devices have been designed, fabricated and evaluated for carrying out the individual processing steps listed above, work toward building fully integrated microfluidic systems for protein analysis has yet to be realized. In this chapter, information will be provided on the nature of proteomic analysis in terms of the challenges associated with the sample type and the microfluidic devices that have been tested to carry out individual processing steps. These include devices such as those for multidimensional electrophoretic separations, solid-phase enzymatic digestions, and solid-phase extractions, all of which have used microfluidics as the functional platform for their implementation. This will be followed by an in-depth review of microfluidic systems, which are defined as units possessing two or more devices assembled into autonomous systems for proteome processing. In addition, information will be provided on the challenges involved in integrating processing steps into a functional system and the approaches adopted for device

  5. Protein patterns of black fungi under simulated Mars-like conditions.

    PubMed

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-29

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  6. Protein patterns of black fungi under simulated Mars-like conditions

    PubMed Central

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-01-01

    Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure. PMID:24870977

  7. Electroporation of cells in microfluidic droplets.

    PubMed

    Zhan, Yihong; Wang, Jun; Bao, Ning; Lu, Chang

    2009-03-01

    Droplet-based microfluidics has raised a lot of interest recently due to its wide applications to screening biological/chemical assays with high throughput. Despite the advances on droplet-based assays involving cells, gene delivery methods that are compatible with the droplet platform have been lacking. In this report, we demonstrate a simple microfluidic device that encapsulates cells into aqueous droplets and then electroporates the encapsulated cells. The electroporation occurs when the cell-containing droplets (in oil) flow through a pair of microelectrodes with a constant voltage established in between. We investigate the parameters and characteristics of the electroporation. We demonstrate delivering enhanced green fluorescent protein (EGFP) plasmid into Chinese hamster ovary (CHO) cells. We envision the application of this technique to high-throughput functional genomics studies based on droplet microfluidics.

  8. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels

    NASA Astrophysics Data System (ADS)

    Deforest, Cole A.; Tirrell, David A.

    2015-05-01

    Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

  9. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

    PubMed Central

    Urbanus, Susan L; de Folter, Stefan; Shchennikova, Anna V; Kaufmann, Kerstin; Immink, Richard GH; Angenent, Gerco C

    2009-01-01

    Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during

  10. Quantity of dietary protein intake, but not pattern of intake, affects net protein balance primarily through differences in protein synthesis in older adults.

    PubMed

    Kim, Il-Young; Schutzler, Scott; Schrader, Amy; Spencer, Horace; Kortebein, Patrick; Deutz, Nicolaas E P; Wolfe, Robert R; Ferrando, Arny A

    2015-01-01

    To examine whole body protein turnover and muscle protein fractional synthesis rate (MPS) following ingestions of protein in mixed meals at two doses of protein and two intake patterns, 20 healthy older adult subjects (52-75 yr) participated in one of four groups in a randomized clinical trial: a level of protein intake of 0.8 g (1RDA) or 1.5 g·kg(-1)·day(-1) (∼2RDA) with uneven (U: 15/20/65%) or even distribution (E: 33/33/33%) patterns of intake for breakfast, lunch, and dinner over the day (1RDA-U, 1RDA-E, 2RDA-U, or 2RDA-E). Subjects were studied with primed continuous infusions of L-[(2)H5]phenylalanine and L-[(2)H2]tyrosine on day 4 following 3 days of diet habituation. Whole body protein kinetics [protein synthesis (PS), breakdown, and net balance (NB)] were expressed as changes from the fasted to the fed states. Positive NB was achieved at both protein levels, but NB was greater in 2RDA vs. 1RDA (94.8 ± 6.0 vs. 58.9 ± 4.9 g protein/750 min; P = 0.0001), without effects of distribution on NB. The greater NB was due to the higher PS with 2RDA vs. 1RDA (15.4 ± 4.8 vs. -18.0 ± 8.4 g protein/750 min; P = 0.0018). Consistent with PS, MPS was greater with 2RDA vs. 1RDA, regardless of distribution patterns. In conclusion, whole body net protein balance was greater with protein intake above recommended dietary allowance (0.8 g protein·kg(-1)·day(-1)) in the context of mixed meals, without demonstrated effects of protein intake pattern, primarily through higher rates of protein synthesis at whole body and muscle levels.

  11. Cell manipulation in microfluidics.

    PubMed

    Yun, Hoyoung; Kim, Kisoo; Lee, Won Gu

    2013-06-01

    Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.

  12. Microfluidic multiplexing in bioanalyses.

    PubMed

    Araz, M Kursad; Tentori, Augusto M; Herr, Amy E

    2013-10-01

    The importance of biological assays spans from clinical diagnostics to environmental monitoring. Simultaneous detection of multiple analytes enhances the efficacy of bioassays by providing more data per assay under standardized conditions. Nevertheless, simultaneous handling and assaying of multiple samples, targets, and experimental conditions can be laborious, reagent consuming, and time intensive. Given these demands, microfluidic platforms have emerged over the past two decades as well-suited approaches for multiplexed assays. Microfluidic design supports integration of assay steps and reproducible sample manipulation across large sets of conditions--all relevant to multiplexed assays. Taken together, reduced reagent consumption, faster assay times, and potential for automation stemming from microfluidic assay design are attractive and needed multiplexed assay performance attributes. This review highlights recent advances in multiplexed bioanalyses benefitting from microfluidic integration.

  13. Microfluidic chemical reaction circuits

    SciTech Connect

    Lee, Chung-cheng; Sui, Guodong; Elizarov, Arkadij; Kolb, Hartmuth C; Huang, Jiang; Heath, James R; Phelps, Michael E; Quake, Stephen R; Tseng, Hsian-rong; Wyatt, Paul; Daridon, Antoine

    2012-06-26

    New microfluidic devices, useful for carrying out chemical reactions, are provided. The devices are adapted for on-chip solvent exchange, chemical processes requiring multiple chemical reactions, and rapid concentration of reagents.

  14. Microfluidics in inorganic chemistry.

    PubMed

    Abou-Hassan, Ali; Sandre, Olivier; Cabuil, Valérie

    2010-08-23

    The application of microfluidics in chemistry has gained significant importance in the recent years. Miniaturized chemistry platforms provide controlled fluid transport, rapid chemical reactions, and cost-saving advantages over conventional reactors. The advantages of microfluidics have been clearly established in the field of analytical and bioanalytical sciences and in the field of organic synthesis. It is less true in the field of inorganic chemistry and materials science; however in inorganic chemistry it has mostly been used for the separation and selective extraction of metal ions. Microfluidics has been used in materials science mainly for the improvement of nanoparticle synthesis, namely metal, metal oxide, and semiconductor nanoparticles. Microfluidic devices can also be used for the formulation of more advanced and sophisticated inorganic materials or hybrids.

  15. Comparative SDS-page protein patterns of four ascaridid nematodes.

    PubMed

    Ashour, A A; Taha, H A; Mohammad A el-H

    1995-12-01

    In order to investigate the degree of homogeneity and heterogeneity of the ascaridid nematodes. Toxascaris leonina, Parascaris equorum, Toxocara canis and T. vitulorum, protein extracts from adult worms of the four nematodes were resolved into a number of bands. Comparative analysis of dominant bands showed that 13 bands were common among the four species, but certain unique bands were also found in each species including 4 in T. vitulorum, one in T. leonina, two in T. canis, while P. equorum shares both T. canis and T. leonina in most of their bands. Among the four ascaridid studied, T. vitulorum appears to be the most divergent species.

  16. High-quality combinatorial protein libraries using the binary patterning approach.

    PubMed

    Bradley, Luke H

    2014-01-01

    Protein combinatorial libraries have become a platform technology for exploring protein sequence space for novel molecules for use in research, synthetic biology, biotechnology, and medicine. To expedite the isolation of proteins with novel/desired functions using screens and selections, high-quality approaches that generate protein libraries rich in folded and soluble structures are desirable for this goal. The binary patterning approach is a protein library design method that incorporates elements of both rational design and combinatorial diversity to specify the arrangement of polar and nonpolar amino acid residues in the context of a desired, folded tertiary structure template. An overview of the considerations necessary to design and construct binary patterned libraries of de novo and natural proteins is presented.

  17. Microfluidic parallel circuit for measurement of hydraulic resistance.

    PubMed

    Choi, Sungyoung; Lee, Myung Gwon; Park, Je-Kyun

    2010-08-31

    We present a microfluidic parallel circuit that directly compares the test channel of an unknown hydraulic resistance with the reference channel with a known resistance, thereby measuring the unknown resistance without any measurement setup, such as standard pressure gauges. Many of microfluidic applications require the precise transport of fluid along a channel network with complex patterns. Therefore, it is important to accurately characterize and measure the hydraulic resistance of each channel segment, and determines whether the device principle works well. However, there is no fluidic device that includes features, such as the ability to diagnose microfluidic problems by measuring the hydraulic resistance of a microfluidic component in microscales. To address the above need, we demonstrate a simple strategy to measure an unknown hydraulic resistance, by characterizing the hydraulic resistance of microchannels with different widths and defining an equivalent linear channel of a microchannel with repeated patterns of a sudden contraction and expansion.

  18. Flock-based microfluidics.

    PubMed

    Hitzbleck, Martina; Lovchik, Robert D; Delamarche, Emmanuel

    2013-05-21

    Flock-based microfluidics are created by depositing hydrophilic microfibers on an adhesive-coated substrate using an electric field. This enables the fabrication of self-powered microfluidics from one or more different kinds of fibers that form 2D and 3D flowpaths, which can wick 40 microliters of liquid per square centimeter. With this approach, large areas of functional wicking materials can be produced at extremely low cost.

  19. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    PubMed

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  20. [Analysis of gingival crevicular fluid. Relation of isoelectric focusing protein patterns to clinical evaluation].

    PubMed

    Aoki, Y; Yoshinaga, E; Tamazawa, O

    1988-12-01

    The purpose of this study was to determine the protein patterns in gingival crevicular fluid relation to the isoelectric focusing protein patterns of GCF and to clinical evaluations. GCF was collected with filter paper from 105 subjects. The probing depth, the gingival index (Löe & Silness) and the plaque index (Silness & Löe) as clinical evaluations The results follow: 1. The main isoelectric focusing protein patterns of GCF were between pH 5.5 and 7.5. In comparison, the GCF and the serum from the same patients showed patterns to similar serum albumin. 2. Between of GCF pH 5.5 and 7.5 the protein patterns that ranged over 60% was pI 5.65, 6.45, 6.55, 6.75 and 7.00. The frequencies of the ranges of protein patterns and clinical evaluation were compared by the X2 test. pI 5.65, 6.45, 6.55 and 6.75 and PD were significant different, as were pI 6.45, 6.55 and 6.75 and GI. But each pI and PIl. were not significantly different.

  1. Extracellular matrix protein patterns guide human chondrocytes adhesion and alignment characterized by vimentin and matrilin-3.

    PubMed

    Pan, Chang-Jiang; Ding, Hong-Yan; Dong, Yun-Xiao

    2013-02-01

    The main purpose of the present study is to investigate the influences of collagen VI (col-VI) patterns on human chondrocytes behaviors. To this end, col-VI stripes with varying width and interstripe spacing are created on polystyrene (PS) surfaces by microcontact printing (μCP). Human chondrocytes are then seeded on these protein patterns and the cell adhesion and alignment are investigated by staining the vimentin and matrilin-3 secreted by seeded chondrocytes. The results indicate that the cells preferentially attach onto the protein areas, rendering cell patterns and the elongated cell shapes. The pattern dimensions can significantly influence cell adhesion, spreading and orientation. The stripe protein patterns can guide cell adhesion and alignment. The cell morphologies can be controlled by carefully designing the pattern shapes and sizes. Our results suggest that the protein patterns can be used to modify biomaterials' surfaces for selective cell-binding and cell alignment. It could provide some cues for the development of novel implantable biomaterials, such as tissue-engineered scaffolds for cartilage replacement, where specific cell alignment is needed.

  2. Protein patterning utilizing region-specific control of wettability by surface modification under atmospheric pressure

    NASA Astrophysics Data System (ADS)

    Lee, Donghee; Kwon, Min-Sung; Hyun, Ji-Chul; Jun, Chang-Duk; Chung, Euiheon; Yang, Sung

    2013-09-01

    Wettability control can be crucial in improving the uniformity of selective protein immobilization in high-density microarrays. In this study, we propose an atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD)-based method in conjunction with photolithography to implement region-specific control of wettability on Si substrate. The proposed PECVD method under atmospheric pressure condition would be a useful alternative of conventional reactive plasma-based treatments methods requiring vacuum condition for uniform protein patterning. Layers with dissimilar wettability and roughness prepared by AP-PECVD process using tetraethoxysilane (TEOS) or TEOS-O2 as precursors could realize uniform protein patterning in a micrometer-scale.

  3. MEMS in microfluidic channels.

    SciTech Connect

    Ashby, Carol Iris Hill; Okandan, Murat; Michalske, Terry A.; Sounart, Thomas L.; Matzke, Carolyn M.

    2004-03-01

    Microelectromechanical systems (MEMS) comprise a new class of devices that include various forms of sensors and actuators. Recent studies have shown that microscale cantilever structures are able to detect a wide range of chemicals, biomolecules or even single bacterial cells. In this approach, cantilever deflection replaces optical fluorescence detection thereby eliminating complex chemical tagging steps that are difficult to achieve with chip-based architectures. A key challenge to utilizing this new detection scheme is the incorporation of functionalized MEMS structures within complex microfluidic channel architectures. The ability to accomplish this integration is currently limited by the processing approaches used to seal lids on pre-etched microfluidic channels. This report describes Sandia's first construction of MEMS instrumented microfluidic chips, which were fabricated by combining our leading capabilities in MEMS processing with our low-temperature photolithographic method for fabricating microfluidic channels. We have explored in-situ cantilevers and other similar passive MEMS devices as a new approach to directly sense fluid transport, and have successfully monitored local flow rates and viscosities within microfluidic channels. Actuated MEMS structures have also been incorporated into microfluidic channels, and the electrical requirements for actuation in liquids have been quantified with an elegant theory. Electrostatic actuation in water has been accomplished, and a novel technique for monitoring local electrical conductivities has been invented.

  4. Global Patterns of Protein Domain Gain and Loss in Superkingdoms

    PubMed Central

    Nasir, Arshan; Kim, Kyung Mo; Caetano-Anollés, Gustavo

    2014-01-01

    Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea. PMID:24499935

  5. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  6. Microfluidic probe: a new tool for integrating microfluidic environments and electronic wafer-probing.

    PubMed

    Routenberg, David A; Reed, Mark A

    2010-01-07

    We demonstrate a new tool for integrating microfluidic channels with commonly used electronic probing techniques. The "microfluidic probe" allows rapid and repeatable fluidic and electronic addressing of small die sites on a variety of substrate types without the need for permanent modification or dicing of the device wafers. We also use the probe to demonstrate locally patterned chemical modification of a substrate. The probes are easily fabricated using standard soft-lithography and basic machining making this a widely accessible technique for electronics and fluidics researchers.

  7. Hybrid microfluidic systems: combining a polymer microfluidic toolbox with biosensors

    NASA Astrophysics Data System (ADS)

    Gärtner, Claudia; Kirsch, Stefanie; Anton, Birgit; Becker, Holger

    2007-01-01

    In this paper we present polymer based microfluidic chips which contain functional elements (electrodes, biosensors) made out of a different material (metals, silicon, organic semiconductors). These hybrid microfluidic devices allow the integration of additional functionality other than the simple manipulation of liquids in the chip and have been developed as a reaction to the increasing requirement for functional integration in microfluidics.

  8. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein

    SciTech Connect

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-09-05

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M–C4BP interaction, and also inform a path towards vaccine design.

  9. Punch card programmable microfluidics.

    PubMed

    Korir, George; Prakash, Manu

    2015-01-01

    Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word "PUNCHCARD MICROFLUIDICS" using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world.

  10. Biopolymers Confined in Surface-Modified Silicon Microfluidic Channels

    NASA Astrophysics Data System (ADS)

    Li, Y.; Pfohl, T.; Yasa, M.; Safinya, C. R.; Kim, J. H.; Kim, M. W.; Wen, Z.

    2001-03-01

    We have developed surface modification techniques for control of wettability and surface charge in lithographically fabricated Si microfluidic channels. Surface microstructures (patterns) with contrasting wetting properties were created using a combination of microcontact printing and polyelectrolyte adsorption. The selective control of the surface property enabled us to devise various techniques for loading and processing biomaterials in the channels. Using fluorescence and laser scanning confocal microscopy, we studied the structure of biopolymers including DNA, F-Actin and microtubules confined in the surface-modified microchannels. The polymers were observed to align linearly along the channels, which suggests that the channel arrays can be used as effective substrates for aligning filamentous proteins for structural characterization by x-ray diffraction. (Work supported by NSF-DMR-9972246, NSF-DMR-0076357, ONR-N00014-00-1-0214, UC-Biotech 99-14, and CULAR 99-216)

  11. Hybrid IC / Microfluidic Chips for the Manipulation of Biological Cells

    NASA Astrophysics Data System (ADS)

    Lee, Hakho

    2005-03-01

    A hybrid IC / Microfluidic chip that can manipulate individual biological cells in a fluid with microscopic resolution has been demonstrated. The chip starts with a custom-designed silicon integrated circuit (IC) produced in a foundry using standard processing techniques. A microfluidic chamber is then fabricated on top of the IC to provide a biocompatible environment. The motion of biological cells in the chamber is controlled using a two-dimensional array of micro-scale electromagnets in the IC that generate spatially patterned magnetic fields. A local peak in the magnetic field amplitude will trap a magnetic bead and an attached cell; by moving the peak's location, the bead-bound cell can be moved to any position on the chip surface above the array. By generating multiple peaks, many cells can be moved independently along separate paths, allowing many different manipulations of individual cells. The hybrid IC / Microfluidic chip can be used, for example, to sort cells or to assemble tissue on micrometer length scales. To prove the concept, an IC / Microfluidic chip was fabricated, based on a custom-designed IC that contained a two-dimensional microcoil array with integrated current sources and control circuits. The chip was tested by trapping and moving biological cells tagged with magnetic beads inside the microfluidic chamber over the array. By combining the power of silicon technology with the biocompatibility of microfluidics, IC / Microfluidic chips will make new types of investigations possible in biological and biomedical studies.

  12. Structural Determination of Biomolecules in Microfluidic Systems

    NASA Astrophysics Data System (ADS)

    Butler, John C.; Menard, Etienne; Rogers, John A.; Wong, Gerard C. L.

    2004-03-01

    Supramolecular biological complexes are often too large to be crystallized for structural studies. Here, we explore the use of microfluidic arrays to order a model self-assembled cytoskeletal system. Filamentous actin (F-actin) is a negatively charged protein rod and is a key structural component in the eukaryotic cytoskeleton. In this context, F-actin can self-assemble with actin binding proteins (ABP) in a highly regulated manner to dynamically form structures for a wide range of biomechanical functions. In this work, we will systematically study the action of 3 types of actin binding proteins (a-actinin, fimbrin, cofilin) on the self-assembled structures of F-actin that have been aligned in microfluidic arrays.

  13. Integrated microfluidic probe station

    NASA Astrophysics Data System (ADS)

    Perrault, C. M.; Qasaimeh, M. A.; Brastaviceanu, T.; Anderson, K.; Kabakibo, Y.; Juncker, D.

    2010-11-01

    The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution—thus hydrodynamically confining the microjet—and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

  14. Integrated microfluidic probe station.

    PubMed

    Perrault, C M; Qasaimeh, M A; Brastaviceanu, T; Anderson, K; Kabakibo, Y; Juncker, D

    2010-11-01

    The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

  15. An ultra-sensitive microfluidic immunoassay using living radical polymerization and porous polymer monoliths.

    SciTech Connect

    Abhyankar, Vinay V.; Singh, Anup K.; Hatch, Anson V.

    2010-07-01

    We present a platform that combines patterned photopolymerized polymer monoliths with living radical polymerization (LRP) to develop a low cost microfluidic based immunoassay capable of sensitive (low to sub pM) and rapid (<30 minute) detection of protein in 100 {micro}L sample. The introduction of LRP functionality to the porous monolith allows one step grafting of functionalized affinity probes from the monolith surface while the composition of the hydrophilic graft chain reduces non-specific interactions and helps to significantly improve the limit of detection.

  16. Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

    PubMed

    Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

    2013-04-05

    To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

  17. Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii.

    PubMed Central

    Silva, A M; Maia, J C; Juliani, M H

    1987-01-01

    Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Images PMID:3571161

  18. Inverse immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human bone marrow megakaryocytes.

    PubMed

    de Larouzière, V; Brouland, J P; Souni, F; Drouet, L; Cramer, E

    1998-06-01

    The time of appearance and pattern of expression of several alpha-granule proteins, von Willebrand factor (VWF), fibrinogen and immunoglobulins (Ig) were examined and compared in human bone marrow megakaryocytes (MK) using an immunocytochemical approach. VWF is synthesized by immature MK, whereas it has been shown that fibrinogen is incorporated from the plasma into alpha-granules. The present study was undertaken in order to determine whether there are chronological and morphological differences in the expression of VWF and fibrinogen in vivo in human MK. Seven paraffin-embedded biopsies of normal human bone marrow were labelled with specific antibodies for VWF and for fibrinogen, detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. and analysed by immunomorphometry. We found a clear, statistically significant. difference in the labelling pattern of VWF and fibrinogen. The expression of other endocytosed alpha-granule proteins, immunoglobulins G and A, was therefore studied in bone marrow MK from two patients with multiple myeloma, one with monoclonal IgG and one with monoclonal IgA. The immunostaining pattern was similar to that of fibrinogen and different from VWF, and characteristic of endocytosed alpha-granule proteins. This study demonstrates that: (i) immunohistochemical staining of MK alpha-granules proteins distinguishes the peripheral cockade distribution pattern of endocytosed protein from the perinuclear pattern of endogenously synthesized proteins; (ii) VWF is present in human bone marrow MK when fibrinogen is not yet detectable: (iii) VWF synthesis ceases while fibrinogen is still being incorporated: (iv) immunoglobulins can be detected in MK cytoplasm, with a staining pattern resembling that of fibrinogen.

  19. Tuning Fluidic Resistance via Liquid Crystal Microfluidics

    PubMed Central

    Sengupta, Anupam

    2013-01-01

    Flow of molecularly ordered fluids, like liquid crystals, is inherently coupled with the average local orientation of the molecules, or the director. The anisotropic coupling—typically absent in isotropic fluids—bestows unique functionalities to the flowing matrix. In this work, we harness this anisotropy to pattern different pathways to tunable fluidic resistance within microfluidic devices. We use a nematic liquid crystalline material flowing in microchannels to demonstrate passive and active modulation of the flow resistance. While appropriate surface anchoring conditions—which imprint distinct fluidic resistances within microchannels under similar hydrodynamic parameters—act as passive cues, an external field, e.g., temperature, is used to actively modulate the flow resistance in the microfluidic device. We apply this simple concept to fabricate basic fluidic circuits, which can be hierarchically extended to create complex resistance networks, without any additional design or morphological patterning of the microchannels. PMID:24256819

  20. Inference of Hopfield-Potts patterns from covariation in protein families: calculation and statistical error bars

    NASA Astrophysics Data System (ADS)

    Cocco, Simona; Monasson, Rémi; Weigt, Martin

    2013-12-01

    We consider the Hopfield-Potts model for the covariation between residues in protein families recently introduced in Cocco, Monasson, Weigt (2013). The patterns of the model are inferred from the data within a new gauge, more symmetric in the residues. We compute the statistical error bars on the pattern components. Results are illustrated on real data for a response regulator receiver domain (Pfam ID PF00072) family.

  1. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals. [Hibiscus rosa-sinensis

    SciTech Connect

    Woodson, W.R.; Handa, A.K.

    1986-04-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of /sup 3/H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide.

  2. How Surface Heterogeneity Affects Protein Adsorption: Annealing of OTS Patterns and Albumin Adsorption Kinetics*

    PubMed Central

    Hodgkinson, Gerald N.; Hlady, Vladimir

    2009-01-01

    Fluorescence microscopy and intensity histogram analysis techniques were used to monitor spatially-resolved albumin adsorption kinetics to model heterogeneous surfaces on sub-μm scales. Several distinct protein subpopulations were resolved, each represented by a normal distribution of adsorption densities on the adsorbent surface. Histogram analyses provided dynamic information of mean adsorption density, spread in adsorption density, and surface area coverage for each distinct protein subpopulation. A simple adsorption model is proposed in which individual protein binding events are predicted by the summation of multiple protein's surface sub-site interactions with different binding energy sub-sites on adsorbent surfaces. This model is predictive of the albumin adsorption on the patterns produced by one step μ-contact printing (μCP) of octadecyltrichlorosilane (OTS) on glass but fails to describe adsorption once the same patterns are altered by a thermal annealing step. PMID:19746205

  3. Microfluidic platforms for mechanobiology

    PubMed Central

    Polacheck, William J.; Li, Ran; Uzel, Sebastien G. M.

    2013-01-01

    Mechanotransduction has been a topic of considerable interest since early studies demonstrated a link between mechanical force and biological response. Until recently, studies of fundamental phenomena were based either on in vivo experiments with limited control or direct access, or on large-scale in vitro studies lacking many of the potentially important physiological factors. With the advent of microfluidics, many of the previous limitations of in vitro testing were eliminated or reduced through greater control or combined functionalities. At the same time, imaging capabilities were tremendously enhanced. In this review, we discuss how microfluidics has transformed the study of mechanotransduction. This is done in the context of the various cell types that exhibit force-induced responses and the new biological insights that have been elucidated. We also discuss new microfluidic studies that could produce even more realistic models of in vivo conditions by combining multiple stimuli or creating a more realistic microenvironment. PMID:23649165

  4. Microfluidic Mixing: A Review

    PubMed Central

    Lee, Chia-Yen; Chang, Chin-Lung; Wang, Yao-Nan; Fu, Lung-Ming

    2011-01-01

    The aim of microfluidic mixing is to achieve a thorough and rapid mixing of multiple samples in microscale devices. In such devices, sample mixing is essentially achieved by enhancing the diffusion effect between the different species flows. Broadly speaking, microfluidic mixing schemes can be categorized as either “active”, where an external energy force is applied to perturb the sample species, or “passive”, where the contact area and contact time of the species samples are increased through specially-designed microchannel configurations. Many mixers have been proposed to facilitate this task over the past 10 years. Accordingly, this paper commences by providing a high level overview of the field of microfluidic mixing devices before describing some of the more significant proposals for active and passive mixers. PMID:21686184

  5. Rapid prototyping of multiphase microfluidics with robotic cutters

    NASA Astrophysics Data System (ADS)

    Li, Zidong; Zhao, Zhengtuo; Lo, Joe Fu-jiou

    2014-03-01

    Microfluidic devices offer novel techniques to address biological and biomedical issues. Standard microfluidic fabrication uses photolithography to pattern channels on silicon wafers with high resolution. Even the relatively straightforward SU8 and soft lithography in microfluidics require investing and training in photolithography, which is also time consuming due to complicated thick resist procedures, including sensitive substrate pretreatment, coating, soft bake, expose, post-exposure bake, and developing steps. However, for applications where low resolution (>200 μm) and high turn-around (> 4 designs/day) prototyping are met with little or no lithography infrastructure, robotic cutters [1] offer flexible options for making glass and PDMS microfluidics. We describe the use of robotics cutters for designing microfluidic geometries, and compliment it with safe glass etching, with depths down to 60 μm. Soft lithography patterning of 200 μm thick PDMS membrane was also explored. Without high equipment investment and lengthy student training, both glass and PDMS microfluidics can be achieved in small facilities using this technique.

  6. Pattern recognition of the secondary structure of proteins (alpha-helix and beta-structure).

    PubMed

    Tohá, J C; Soto, M A; Chinga, H

    1990-09-21

    In this paper, an algorithm for the pattern recognition of secondary structure of proteins is proposed. The procedure simultaneously evaluates the contribution of all the residues of a given peptide to its conformation. By means of the algorithm it is possible to select from a universe of well known proteins the most representative alpha-helix and beta-structure peptides, and to use these peptides, as screening matrices to define the unknown structure of any peptide.

  7. The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

    PubMed Central

    Slabodnick, Mark M.; Ruby, J. Graham; Dunn, Joshua G.; Feldman, Jessica L.; DeRisi, Joseph L.; Marshall, Wallace F.

    2014-01-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system. PMID:24823688

  8. The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

    PubMed

    Slabodnick, Mark M; Ruby, J Graham; Dunn, Joshua G; Feldman, Jessica L; DeRisi, Joseph L; Marshall, Wallace F

    2014-05-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.

  9. Punch Card Programmable Microfluidics

    PubMed Central

    Korir, George; Prakash, Manu

    2015-01-01

    Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word “PUNCHCARD MICROFLUIDICS” using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world. PMID:25738834

  10. Establishing knowledge on the sequence arrangement pattern of nucleated protein folding

    PubMed Central

    Leng, Fei; Xu, Chao; Xia, Xia-Yu; Pan, Xian-Ming

    2017-01-01

    The heat-tolerance mechanisms of (hyper)thermophilic proteins provide a unique opportunity to investigate the unsolved protein folding problem. In an attempt to determine whether the interval between residues in sequence might play a role in determining thermostability, we constructed a sequence interval-dependent value function to calculate the residue pair frequency. Additionally, we identified a new sequence arrangement pattern, where like-charged residues tend to be adjacently assembled, while unlike-charged residues are distributed over longer intervals, using statistical analysis of a large sequence database. This finding indicated that increasing the intervals between unlike-charged residues can increase protein thermostability, with the arrangement patterns of these charged residues serving as thermodynamically favorable nucleation points for protein folding. Additionally, we identified that the residue pairs K-E, R-E, L-V and V-V involving long sequence intervals play important roles involving increased protein thermostability. This work demonstrated a novel approach for considering sequence intervals as keys to understanding protein folding. Our findings of novel relationships between residue arrangement and protein thermostability can be used in industry and academia to aid the design of thermostable proteins. PMID:28273143

  11. Development & Characterization of Multifunctional Microfluidic Materials

    NASA Astrophysics Data System (ADS)

    Ucar, Ahmet Burak

    developing 'smart' windows and heat management. To better design new color changing elastomers, we investigated the role of the network geometry on liquid replacement efficiency with the aid of a multiphysics modeling and simulation software package, COMSOL. We simulated the liquid flow in various network geometries. Serpentine, parallel channel and lattice networks, as well as their tapered versions were compared. The comparison criteria were based on rapid and uniform liquid replacement with the least amount of dye/liquid required, for which we set multiple constraints such as constant inlet pressure or total channel area. We demonstrated that the tapered lattice type network provided the most rapid and uniform replacement with minimal liquid waste. Next, we designed a simple and inexpensive liquid dispensing microfluidic material which does not require complex micromachining techniques or automated actuators. It consisted of only a PDMS matrix with embedded chambers and channels. 'Pores/slits' were made on the surface and the liquid was released by contact on the dispensing surface of the material. We varied the network design, geometry, dimension, slit shape and length, and tested the material's liquid release performance. Promising preliminary results were obtained but for an end product with repeatable and reproducible performance, both material fabrication and characterization need to be improved further. Finally, we describe an alternative material/method for the fabrication of microfluidic materials. We aimed to replace the conventional fabrication material PDMS with Polyethylene (PE) sheets. The sheets were as transparent and flexible as PDMS, and also thinner. Channel patterns were drawn with a polymer solution of PolyVinylAlcohol (PVA), which is immiscible with PE, and captured in between the two PE sheets. After fusing the PE sheets on a hot press, PVA was washed off with water, so that the 'microfluidic channels' were successfully created. The produced channel

  12. Microfluidic Flame Barrier

    NASA Technical Reports Server (NTRS)

    Mungas, Gregory S. (Inventor); Fisher, David J. (Inventor); Mungas, Christopher (Inventor)

    2013-01-01

    Propellants flow through specialized mechanical hardware that is designed for effective and safe ignition and sustained combustion of the propellants. By integrating a micro-fluidic porous media element between a propellant feed source and the combustion chamber, an effective and reliable propellant injector head may be implemented that is capable of withstanding transient combustion and detonation waves that commonly occur during an ignition event. The micro-fluidic porous media element is of specified porosity or porosity gradient selected to be appropriate for a given propellant. Additionally the propellant injector head design integrates a spark ignition mechanism that withstands extremely hot running conditions without noticeable spark mechanism degradation.

  13. Experimental Microfluidic System

    NASA Technical Reports Server (NTRS)

    Culbertson, Christopher; Gonda, Steve; Ramsey, John Michael

    2005-01-01

    The ultimate goal of this project is to integrate microfluidic devices with NASA's space bioreactor systems. In such a system, the microfluidic device would provide realtime feedback control of the bioreactor by monitoring pH, glucose, and lactate levels in the cell media; and would provide an analytical capability to the bioreactor in exterrestrial environments for monitoring bioengineered cell products and health changes in cells due to environmental stressors. Such integrated systems could be used as biosentinels both in space and on planet surfaces. The objective is to demonstrate the ability of microfabricated devices to repeatedly and reproducibly perform bead cytometry experiments in micro, lunar, martian, and hypergravity (1.8g).

  14. In vitro evaluation and in vivo demonstration of a biomimetic, hemocompatible, microfluidic artificial lung.

    PubMed

    Kovach, K M; LaBarbera, M A; Moyer, M C; Cmolik, B L; van Lunteren, E; Sen Gupta, A; Capadona, J R; Potkay, J A

    2015-03-07

    Despite the promising potential of microfluidic artificial lungs, current designs suffer from short functional lifetimes due to surface chemistry and blood flow patterns that act to reduce hemocompatibility. Here, we present the first microfluidic artificial lung featuring a hemocompatible surface coating and a biomimetic blood path. The polyethylene-glycol (PEG) coated microfluidic lung exhibited a significantly improved in vitro lifetime compared to uncoated controls as well as consistent and significantly improved gas exchange over the entire testing period. Enabled by our hemocompatible PEG coating, we additionally describe the first extended (3 h) in vivo demonstration of a microfluidic artificial lung.

  15. A "place n play" modular pump for portable microfluidic applications.

    PubMed

    Li, Gang; Luo, Yahui; Chen, Qiang; Liao, Lingying; Zhao, Jianlong

    2012-03-01

    This paper presents an easy-to-use, power-free, and modular pump for portable microfluidic applications. The pump module is a degassed particle desorption polydimethylsiloxane (PDMS) slab with an integrated mesh-shaped chamber, which can be attached on the outlet port of microfluidic device to absorb the air in the microfluidic system and then to create a negative pressure for driving fluid. Different from the existing monolithic degassed PDMS pumps that are generally restricted to limited pumping capacity and are only compatible with PDMS-based microfluidic devices, this pump can offer various possible configures of pumping power by varying the geometries of the pump or by combining different pump modules and can also be employed in any material microfluidic devices. The key advantage of this pump is that its operation only requires the user to place the degassed PDMS slab on the outlet ports of microfluidic devices. To help design pumps with a suitable pumping performance, the effect of pump module geometry on its pumping capacity is also investigated. The results indicate that the performance of the degassed PDMS pump is strongly dependent on the surface area of the pump chamber, the exposure area and the volume of the PDMS pump slab. In addition, the initial volume of air in the closed microfluidic system and the cross-linking degree of PDMS also affect the performance of the degassed PDMS pump. Finally, we demonstrated the utility of this modular pumping method by applying it to a glass-based microfluidic device and a PDMS-based protein crystallization microfluidic device.

  16. Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation.

    PubMed

    Salem-Izacc, S M; Jesuino, R S; Brito, W A; Pereira, M; Felipe, M S; Soares, C M

    1997-01-01

    In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

  17. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.A.; Sulkin, S.T.; Lee, F.B.; Henny, C.J.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12?23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23?39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  18. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12-23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23-39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  19. Chemistry in Microfluidic Channels

    ERIC Educational Resources Information Center

    Chia, Matthew C.; Sweeney, Christina M.; Odom, Teri W.

    2011-01-01

    General chemistry introduces principles such as acid-base chemistry, mixing, and precipitation that are usually demonstrated in bulk solutions. In this laboratory experiment, we describe how chemical reactions can be performed in a microfluidic channel to show advanced concepts such as laminar fluid flow and controlled precipitation. Three sets of…

  20. Design and fabrication of polymer-based microfluidic platforms for BioMEMS applications

    NASA Astrophysics Data System (ADS)

    Lai, Siyi

    The goal of this study is to design and fabricate polymer microfluidic devices for BioMEMS applications. The emphasis is on the design of microfluidic functions and the development of a new packaging technique. A microfluidic platform was designed on a compact disk (CD) for medical diagnostics, which includes functions such as pumping, valving, sample/reagent loading, mixing, metering, and separation. The fluid propulsion was based on the centrifugal force. A passive capillary valve, which is based on a pressure barrier that develops when the cross-section of the capillary expands abruptly, was used to control the fluid flow. Micromixing was achieved by impinging mixing and bend-induced vortices. Integration of these microfluidic functions was applied in a two-point calibration system for medical diagnostics and a cascade micromixer for protein reconstitution. A specific application was for enzyme-linked immunosorbent assays (ELISA). It has been demonstrated successfully to realize the necessary microfluidic functions for the ELISA process on a CD. The preliminary analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate, and has advantages such as less reagent consumption and shorter assay time over the conventional one. A new resin-gas injection technique was developed for bonding and surface modification of polymer microfluidic devices. This method can easily bond biochips with complex flow patterns. By adding surface modification agents, the interfacial free energy of the substrate with water can be controlled. Local modification of the channel surface can also be achieved through sequential resin-gas injection in conjunction with the masking technique. For application, this technique was used to form a layer of dry monolithic stationary hydrogel on the walls of a microchannel, serving as a sieving material for electrophoresis separation of DNA

  1. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    SciTech Connect

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-08-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates.

  2. Reversible Dialysis in a Microfluidic Formulator.

    NASA Astrophysics Data System (ADS)

    Selimovic, Seila; Shim, Jung-Uk; Fraden, Seth

    2006-03-01

    In order to facilitate the screening of conditions for protein crystallization, we have been using the Microfluidic Formulator chip (Stephen Quake, PNAS Vol. 101, 40 ). This PDMS device allows us to mix up to 40 different reagents and protein solutions. We use this combinatorial approach along with a ``drop-on-demand'' method whereby we employ on-chip positive displacement pumps to form aqueous droplets containing protein and separate them by plugs of oil. Subsequently, the aqueous drops containing protein are guided by surface tension forces into storage chambers. To control the chemical potential of these sub-nanoliter protein samples, we fabricate reservoirs underneath the storage compartments. A thin PDMS membrane that is permeable to water, but not to protein or salt, separates the reservoirs from the storage chambers. Water can permeate into or out of the stored samples until the chemical potentials of the reservoir and the protein solution are equal leading to protein crystallization in some chambers.

  3. Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

    PubMed Central

    del Val, Coral; White, Stephen H.

    2014-01-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  4. Biophysical Models of Protein Evolution: Understanding the Patterns of Evolutionary Sequence Divergence.

    PubMed

    Echave, Julian; Wilke, Claus O

    2017-03-15

    For decades, rates of protein evolution have been interpreted in terms of the vague concept of functional importance. Slowly evolving proteins or sites within proteins were assumed to be more functionally important and thus subject to stronger selection pressure. More recently, biophysical models of protein evolution, which combine evolutionary theory with protein biophysics, have completely revolutionized our view of the forces that shape sequence divergence. Slowly evolving proteins have been found to evolve slowly because of selection against toxic misfolding and misinteractions, linking their rate of evolution primarily to their abundance. Similarly, most slowly evolving sites in proteins are not directly involved in function, but mutating these sites has a large impact on protein structure and stability. In this article, we review the studies in the emerging field of biophysical protein evolution that have shaped our current understanding of sequence divergence patterns. We also propose future research directions to develop this nascent field. Expected final online publication date for the Annual Review of Biophysics Volume 46 is May 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  5. Microfluidic Sample Preparation for Immunoassays

    SciTech Connect

    Visuri, S; Benett, W; Bettencourt, K; Chang, J; Fisher, K; Hamilton, J; Krulevitch, P; Park, C; Stockton, C; Tarte, L; Wang, A; Wilson, T

    2001-08-09

    Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. to support detection instruments, they are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. They are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Many of these fluidic functions are accomplished with acoustic radiation pressure or dielectrophoresis. They are integrating these technologies into packaged systems with pumps and valves to control fluid flow through the fluidic circuit.

  6. Magnetic digital microfluidics - a review.

    PubMed

    Zhang, Yi; Nguyen, Nam-Trung

    2017-03-14

    A digital microfluidic platform manipulates droplets on an open surface. Magnetic digital microfluidics utilizes magnetic forces for actuation and offers unique advantages compared to other digital microfluidic platforms. First, the magnetic particles used in magnetic digital microfluidics have multiple functions. In addition to serving as actuators, they also provide a functional solid substrate for molecule binding, which enables a wide range of applications in molecular diagnostics and immunodiagnostics. Second, magnetic digital microfluidics can be manually operated in a "power-free" manner, which allows for operation in low-resource environments for point-of-care diagnostics where even batteries are considered a luxury item. This review covers research areas related to magnetic digital microfluidics. This paper first summarizes the current development of magnetic digital microfluidics. Various methods of droplet manipulation using magnetic forces are discussed, ranging from conventional magnetic particle-based actuation to the recent development of ferrofluids and magnetic liquid marbles. This paper also discusses several new approaches that use magnetically controlled flexible substrates for droplet manipulation. In addition, we emphasize applications of magnetic digital microfluidics in biosensing and medical diagnostics, and identify the current limitations of magnetic digital microfluidics. We provide a perspective on possible solutions to close these gaps. Finally, the paper discusses the future improvement of magnetic digital microfluidics to explore potential new research directions.

  7. Oxygen control with microfluidics.

    PubMed

    Brennan, Martin D; Rexius-Hall, Megan L; Elgass, Laura Jane; Eddington, David T

    2014-11-21

    Cellular function and behavior are affected by the partial pressure of O2, or oxygen tension, in the microenvironment. The level of oxygenation is important, as it is a balance of oxygen availability and oxygen consumption that is necessary to maintain normoxia. Changes in oxygen tension, from above physiological oxygen tension (hyperoxia) to below physiological levels (hypoxia) or even complete absence of oxygen (anoxia), trigger potent biological responses. For instance, hypoxia has been shown to support the maintenance and promote proliferation of regenerative stem and progenitor cells. Paradoxically, hypoxia also contributes to the development of pathological conditions including systemic inflammatory response, tumorigenesis, and cardiovascular disease, such as ischemic heart disease and pulmonary hypertension. Current methods to study cellular behavior in low levels of oxygen tension include hypoxia workstations and hypoxia chambers. These culture systems do not provide oxygen gradients that are found in vivo or precise control at the microscale. Microfluidic platforms have been developed to overcome the inherent limits of these current methods, including lack of spatial control, slow equilibration, and unachievable or difficult coupling to live-cell microscopy. The various applications made possible by microfluidic systems are the topic of this review. In order to understand how the microscale can be leveraged for oxygen control of cells and tissues within microfluidic systems, some background understanding of diffusion, solubility, and transport at the microscale will be presented in addition to a discussion on the methods for measuring the oxygen tension in microfluidic channels. Finally the various methods for oxygen control within microfluidic platforms will be discussed including devices that rely on diffusion from liquid or gas, utilizing on-or-off-chip mixers, leveraging cellular oxygen uptake to deplete the oxygen, relying on chemical reactions in

  8. Protein coverage on polymer nanolayers leading to mesenchymal stem cell patterning.

    PubMed

    You, Jungmok; Yoshida, Akihito; Heo, June Seok; Kim, Han-Soo; Kim, Hyun Ok; Tamada, Kaoru; Kim, Eunkyoung

    2011-10-21

    Interactions of gelatin and albumin with a photo-reactive diphenylamino-s-triazine bridged p-phenylene vinylene polymer (DTOPV) were examined by using surface plasmon resonance (SPR) spectroscopy to explore the effect of the polymer structure on protein coverage of DTOPV nanofilms. The SPR data revealed a significant increase of gelatin adsorption on UV-DTOPV nanofilms, while the adsorption of albumin was decreased by UV exposure in the time frame of the experiment. We also found that the selective adsorption of these proteins was highly dependent on the protein concentration; the highest selectivity of protein adsorption was obtained at the lowest concentration (3.5 μg ml(-1)), while no selective adsorption was confirmed at high concentrations (350 and 1000 μg ml(-1)). The selective attachment of mesenchymal stem cells (MSCs) was directly correlated with the selective adsorption of these proteins onto DTOPV nanofilms. The MSCs attachment onto UV-DTOPV films was promoted with only small mass coverage of gelatin, which led to MSC patterning onto the patterned DTOPV nanofilms successfully. The role of cell adhesion proteins that we found in this study will be a clue to elucidate the complex response of biomolecules on functional polymer nanolayers, and contribute to build up biocompatible surfaces on various advanced materials for the sake of cell engineering and medical implants.

  9. Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

    PubMed Central

    Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

    2012-01-01

    Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum. PMID:22862921

  10. Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures.

    PubMed

    Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

    2012-08-01

    Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi -Coniosporium perforans, Exophiala jeanselmei - and of the extremophilic fungus -Friedmanniomyces endolithicus - were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.

  11. Ultrathin coatings from isocyanate terminated star PEG prepolymers: patterning of proteins on the layers.

    PubMed

    Groll, Juergen; Haubensak, Wulf; Ameringer, Thomas; Moeller, Martin

    2005-03-29

    This study presents the easy and fast patterning of low molecular weight molecules that act as binding partners for proteins on Star PEG coatings. These coatings are prepared from isocyanate terminated star shaped prepolymers and form a highly cross-linked network on the substrate in which the stars are connected via urea groups and free amino groups are present. Streptavidin has been patterned on these layers by microcontact printing (muCP) of an amino reactive biotin derivative and consecutive binding of streptavidin to the biotin. Patterns of Ni(2+)-nitriltriacetic acid (NTA) receptors have been prepared by printing amino functional NTA molecules in freshly prepared Star PEG layers that still contain amino reactive isocyanate groups. Complexation of the NTA groups with Ni(II) ions enabled the binding of His-tag enhanced green fluorescent protein (EGFP) in the desired pattern on the substrates. Since the unmodified Star PEG layers prevent unspecific protein adsorption, His-EGFP could selectively be bound to the sample by immersion into crude, nonpurified His-tag EGFP containing cell lysate.

  12. Collective oscillations and coupled modes in confined microfluidic droplet arrays

    NASA Astrophysics Data System (ADS)

    Schiller, Ulf D.; Fleury, Jean-Baptiste; Seemann, Ralf; Gompper, Gerhard

    Microfluidic droplets have a wide range of applications ranging from analytic assays in cellular biology to controlled mixing in chemical engineering. Ensembles of microfluidic droplets are interesting model systems for non-equilibrium many-body phenomena. When flowing in a microchannel, trains of droplets can form microfluidic crystals whose dynamics are governed by long-range hydrodynamic interactions and boundary effects. In this contribution, excitation mechanisms for collective waves in dense and confined microfluidic droplet arrays are investigated by experiments and computer simulations. We demonstrate that distinct modes can be excited by creating specific `defect' patterns in flowing droplet trains. While longitudinal modes exhibit a short-lived cascade of pairs of laterally displacing droplets, transversely excited modes form propagating waves that behave like microfluidic phonons. We show that the confinement induces a coupling between longitudinal and transverse modes. We also investigate the life time of the collective oscillations and discuss possible mechanisms for the onset of instabilities. Our results demonstrate that microfluidic phonons can exhibit effects beyond the linear theory, which can be studied particularly well in dense and confined systems. This work was supported by Deutsche Forschungsgemeinschaft under Grant No. SE 1118/4.

  13. Bonding PMMA microfluidics using commercial microwave ovens

    NASA Astrophysics Data System (ADS)

    Toossi, A.; Moghadas, H.; Daneshmand, M.; Sameoto, D.

    2015-08-01

    In this paper, a novel low-cost, rapid substrate-bonding technique is successfully applied to polymethyl methacrylate (PMMA) microfluidics bonding for the first time. This technique uses a thin intermediate metallic microwave susceptor layer at the interface of the bonding site (microchannels) which produces localized heating required for bonding during microwave irradiation. The metallic susceptor pattern is designed using a multiphysics simulation model developed in ANSYS Multiphysics software (high-frequency structural simulation (HFSS) coupled with ANSYS-Thermal). In our experiments, the required microwave energy for bonding is delivered using a relatively inexpensive, widely accessible commercial microwave oven. Using this technique, simple PMMA microfluidics prototypes are successfully bonded and sealed in less than 35 seconds with a minimum measured bond strength of 1.375 MPa.

  14. Microfluidic systems for electrochemical and biological studies

    SciTech Connect

    Ackler, H., LLNL

    1998-05-01

    Microfluidic devices with microelectrodes have the potential to enable studies of phenomena at size scales where behavior may be dominated by different mechanisms than at macroscales. Through our work developing microfluidic devices for dielectrophoretic separation and sensing of cells and particles, we have fabricated devices from which general or more specialized research devices may be derived. Fluid channels from 80 {micro}m wide X 20 {micro}m deep to 1 mm wide to 200 {micro}m deep have been fabricated in glass, with lithographically patterned electrodes from 10 to 80 {micro}m wide on one or both sides on the channels and over topographies tens of microns in heights. the devices are designed to easily interface to electronic and fluidic interconnect packages that permit reuse of devices, rather than one-time use, crude glue-based methods. Such devices may be useful for many applications of interest to the electrochemical and biological community.

  15. Cell-based bioassays in microfluidic systems

    NASA Astrophysics Data System (ADS)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.

    2004-12-01

    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  16. Microfluidic technologies for studying synthetic circuits.

    PubMed

    Lin, Benjamin; Levchenko, Andre

    2012-08-01

    Advances in synthetic biology have augmented the available toolkit of biomolecular modules, allowing engineering and manipulation of signaling in a variety of organisms, ranging in complexity from single bacteria and eukaryotic cells to multi-cellular systems. The richness of synthetic circuit outputs can be dramatically enhanced by sophisticated environmental control systems designed to precisely pattern spatial-temporally heterogeneous environmental stimuli controlling these circuits. Moreover, the performance of the synthetic modules and 'blocks' needed to assemble more complicated networks requires more complete characterization as a function of arbitrarily complex environmental inputs. Microfluidic technologies are poised to meet these needs through a variety of innovative designs capitalizing on the unique benefits of manipulating fluids on the micro-scales and nano-scales. This review discusses the utility of microfluidics for the study of synthetic circuits and highlights recent work in the area.

  17. Biased inheritance of the protein PatN frees vegetative cells to initiate patterned heterocyst differentiation.

    PubMed

    Risser, Douglas D; Wong, Francis C Y; Meeks, John C

    2012-09-18

    Heterocysts, cells specialized for nitrogen fixation in certain filamentous cyanobacteria, appear singly in a nonrandom spacing pattern along the chain of vegetative cells. A two-stage, biased initiation and competitive resolution model has been proposed to explain the establishment of this spacing pattern. There is substantial evidence that competitive resolution of a subset of cells initiating differentiation occurs by interactions between a self-enhancing activator protein, HetR, and a diffusible pentapeptide inhibitor PatS-5 (RGSGR). Results presented here show that the absence of a unique membrane protein, PatN, in Nostoc punctiforme strain ATCC 29133 leads to a threefold increase in heterocyst frequency and a fourfold decrease in the vegetative cell interval between heterocysts. A PatN-GFP translational fusion shows a pattern of biased inheritance in daughter vegetative cells of ammonium-grown cultures. Inactivation of another heterocyst patterning gene, patA, is epistatic to inactivation of patN, and transcription of patA increases in a patN-deletion strain, implying that patN may function by modulating levels of patA. The presence of PatN is hypothesized to decrease the competency of a vegetative cell to initiate heterocyst differentiation, and the cellular concentration of PatN is dependent on cell division that results in cells transiently depleted of PatN. We suggest that biased inheritance of cell-fate determinants is a phylogenetic domain-spanning paradigm in the development of biological patterns.

  18. Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2014-10-01

    Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

  19. Direct digital manufacturing of autonomous centrifugal microfluidic device

    NASA Astrophysics Data System (ADS)

    Ukita, Yoshiaki; Takamura, Yuzuru; Utsumi, Yuichi

    2016-06-01

    This paper presents strategies that attempt to solve two key problems facing the commercialization of microfluidics: cost reduction in microfluidic chip manufacturing and microfluidic device driver development. To reduce the cost of microfluidic chip manufacturing, we propose to use of three-dimensional (3D) printers for direct digital manufacturing (DDM). An evaluation of 3D micro-scale structure printing using several 3D printers is reported, and some of the technical issues to be addressed in the future are suggested. To evaluate micro-scale printing, three types of 3D printers, with the ability to print structures on the scale of several hundred meters, were selected by first screening six 3D printers. Line and space patterns with line widths of 100-500 µm and an aspect ratio of one were printed and evaluated. The estimated critical dimension was around 200 µm. The manufacturing of a monolithic microfluidic chip with embedded channels was also demonstrated. Monolithic microfluidic chips with embedded microchannels having 500 × 500 and 250 × 250 µm2 cross sections and 2-20 mm lengths were printed, and the fidelity of the channel shape, residual supporting material, and flow of liquid water were evaluated. The liquid flow evaluation showed that liquid water could flow through all of the microchannels with the 500 × 500 µm2 cross section, whereas this was not possible through some of the channels with the 250 × 250 µm2 cross section because of the residual resin or supporting material. To reduce the device-driver cost, we propose to use of the centrifugal microfluidic concept. An autonomous microfluidic device that could implement sequential flow control under a steadily rotating condition was printed. Four-step flow injection under a steadily rotating condition at 1500 rpm was successfully demonstrated without any external triggering such as changing the rotational speed.

  20. Droplet microfluidics based microseparation systems.

    PubMed

    Xiao, Zhiliang; Niu, Menglei; Zhang, Bo

    2012-06-01

    Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.

  1. Graphene-based microfluidics for serial crystallography.

    PubMed

    Sui, Shuo; Wang, Yuxi; Kolewe, Kristopher W; Srajer, Vukica; Henning, Robert; Schiffman, Jessica D; Dimitrakopoulos, Christos; Perry, Sarah L

    2016-08-02

    Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.

  2. Rapid fabrication of supercapacitor electrodes using bionanoscaffolds in capillary microfluidics

    NASA Astrophysics Data System (ADS)

    Zang, F.; Chu, S.; Gerasopoulos, K.; Culver, J. N.; Ghodssi, R.

    2015-12-01

    This paper reports the utilization of capillary microfluidics to rapidly create nanostructure-patterned electrodes for energy storage applications. Using patterned photoresist as open-channel capillary microfluidics, Tobacco mosaic virus (TMV) bio-nanoscaffolds suspended in solution are autonomously delivered onto planar gold electrodes over a 1 cm2 area. The TMVs assemble on the electrode and form a dense bio-nanoscaffold layer due to enhanced evaporation-assisted assembly in the open-channel capillary microfluidic device within an hour. The TMV structures are coated with Ni/NiO through electroless plating and thermal oxidation to form supercapacitor electrodes. The galvanostatic charge/discharge cycle showed a 3.6-fold increase in areal capacitance for the nanostructured electrode compared to planar structures.

  3. Mapping Protein Abundance Patterns in the Brain Using Voxelation Combined with Liquid Chromatography and Mass Spectrometry

    PubMed Central

    Petyuk, Vladislav A.; Qian, Wei-Jun; Smith, Richard D.; Smith, Desmond J.

    2009-01-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps. PMID:19654045

  4. Patterning protein molecules on poly(ethylene glycol) coated Si(111).

    PubMed

    Jun, Yongseok; Cha, Taewoon; Guo, Athena; Zhu, X-Y

    2004-08-01

    We demonstrate spatially localized immobilization of protein molecules on high-density poly(ethylene glycol) (PEG) coated Si(111). Patterns of HO- and CH3O-terminated PEG regions are formed on silicon surfaces based on soft lithography techniques and an efficient reaction between alcohol functional groups and chlorine-terminated silicon. Activation of the HO-terminated PEG brush is achieved via either partial oxidation to form aldehyde groups or via attachment of efficient leaving groups. Protein molecules are covalently immobilized to these activated regions on the PEG/Si surface.

  5. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  6. Centrifugal microfluidic platforms: advanced unit operations and applications.

    PubMed

    Strohmeier, O; Keller, M; Schwemmer, F; Zehnle, S; Mark, D; von Stetten, F; Zengerle, R; Paust, N

    2015-10-07

    Centrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term "process chain" to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as

  7. Microfluidic Electroporation for Cellular Analysis and Delivery

    PubMed Central

    Geng, Tao

    2013-01-01

    Electroporation is a simple yet powerful technique for breaching cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented. PMID:23917998

  8. The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis.

    PubMed

    Houtmeyers, Rob; Souopgui, Jacob; Tejpar, Sabine; Arkell, Ruth

    2013-10-01

    The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.

  9. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    PubMed

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

  10. Automatic classification and pattern discovery in high-throughput protein crystallization trials.

    PubMed

    Cumbaa, Christian; Jurisica, Igor

    2005-01-01

    Conceptually, protein crystallization can be divided into two phases search and optimization. Robotic protein crystallization screening can speed up the search phase, and has a potential to increase process quality. Automated image classification helps to increase throughput and consistently generate objective results. Although the classification accuracy can always be improved, our image analysis system can classify images from 1,536-well plates with high classification accuracy (85%) and ROC score (0.87), as evaluated on 127 human-classified protein screens containing 5,600 crystal images and 189,472 non-crystal images. Data mining can integrate results from high-throughput screens with information about crystallizing conditions, intrinsic protein properties, and results from crystallization optimization. We apply association mining, a data mining approach that identifies frequently occurring patterns among variables and their values. This approach segregates proteins into groups based on how they react in a broad range of conditions, and clusters cocktails to reflect their potential to achieve crystallization. These results may lead to crystallization screen optimization, and reveal associations between protein properties and crystallization conditions. We also postulate that past experience may lead us to the identification of initial conditions favorable to crystallization for novel proteins.

  11. Microfluidic redox battery.

    PubMed

    Lee, Jin Wook; Goulet, Marc-Antoni; Kjeang, Erik

    2013-07-07

    A miniaturized microfluidic battery is proposed, which is the first membraneless redox battery demonstrated to date. This unique concept capitalizes on dual-pass flow-through porous electrodes combined with stratified, co-laminar flow to generate electrical power on-chip. The fluidic design is symmetric to allow for both charging and discharging operations in forward, reverse, and recirculation modes. The proof-of-concept device fabricated using low-cost materials integrated in a microfluidic chip is shown to produce competitive power levels when operated on a vanadium redox electrolyte. A complete charge/discharge cycle is performed to demonstrate its operation as a rechargeable battery, which is an important step towards providing sustainable power to lab-on-a-chip and microelectronic applications.

  12. Microfluidic colloid filtration

    NASA Astrophysics Data System (ADS)

    Linkhorst, John; Beckmann, Torsten; Go, Dennis; Kuehne, Alexander J. C.; Wessling, Matthias

    2016-03-01

    Filtration of natural and colloidal matter is an essential process in today’s water treatment processes. The colloidal matter is retained with the help of micro- and nanoporous synthetic membranes. Colloids are retained in a “cake layer” – often coined fouling layer. Membrane fouling is the most substantial problem in membrane filtration: colloidal and natural matter build-up leads to an increasing resistance and thus decreasing water transport rate through the membrane. Theoretical models exist to describe macroscopically the hydrodynamic resistance of such transport and rejection phenomena; however, visualization of the various phenomena occurring during colloid retention is extremely demanding. Here we present a microfluidics based methodology to follow filter cake build up as well as transport phenomena occuring inside of the fouling layer. The microfluidic colloidal filtration methodology enables the study of complex colloidal jamming, crystallization and melting processes as well as translocation at the single particle level.

  13. Microfluidic channel fabrication method

    DOEpatents

    Arnold, Don W.; Schoeniger, Joseph S.; Cardinale, Gregory F.

    2001-01-01

    A new channel structure for microfluidic systems and process for fabricating this structure. In contrast to the conventional practice of fabricating fluid channels as trenches or grooves in a substrate, fluid channels are fabricated as thin walled raised structures on a substrate. Microfluidic devices produced in accordance with the invention are a hybrid assembly generally consisting of three layers: 1) a substrate that can or cannot be an electrical insulator; 2) a middle layer, that is an electrically conducting material and preferably silicon, forms the channel walls whose height defines the channel height, joined to and extending from the substrate; and 3) a top layer, joined to the top of the channels, that forms a cover for the channels. The channels can be defined by photolithographic techniques and are produced by etching away the material around the channel walls.

  14. Microfluidic binary phase flow

    NASA Astrophysics Data System (ADS)

    Angelescu, Dan; Menetrier, Laure; Wong, Joyce; Tabeling, Patrick; Salamitou, Philippe

    2004-03-01

    We present a novel binary phase flow regime where the two phases differ substantially in both their wetting and viscous properties. Optical tracking particles are used in order to investigate the details of such multiphase flow inside capillary channels. We also describe microfluidic filters we have developed, capable of separating the two phases based on capillary pressure. The performance of the filters in separating oil-water emulsions is discussed. Binary phase flow has been previously used in microchannels in applications such as emulsion generation, enhancement of mixing and assembly of custom colloidal paticles. Such microfluidic systems are increasingly used in a number of applications spanning a diverse range of industries, such as biotech, pharmaceuticals and more recently the oil industry.

  15. Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants.

    PubMed

    Klimmek, Frank; Sjödin, Andreas; Noutsos, Christos; Leister, Dario; Jansson, Stefan

    2006-03-01

    We have analyzed gene regulation of the Lhc supergene family in poplar (Populus spp.) and Arabidopsis (Arabidopsis thaliana) using digital expression profiling. Multivariate analysis of the tissue-specific, environmental, and developmental Lhc expression patterns in Arabidopsis and poplar was employed to characterize four rarely expressed Lhc genes, Lhca5, Lhca6, Lhcb7, and Lhcb4.3. Those genes have high expression levels under different conditions and in different tissues than the abundantly expressed Lhca1 to 4 and Lhcb1 to 6 genes that code for the 10 major types of higher plant light-harvesting proteins. However, in some of the datasets analyzed, the Lhcb4 and Lhcb6 genes as well as an Arabidopsis gene not present in poplar (Lhcb2.3) exhibited minor differences to the main cooperative Lhc gene expression pattern. The pattern of the rarely expressed Lhc genes was always found to be more similar to that of PsbS and the various light-harvesting-like genes, which might indicate distinct physiological functions for the rarely and abundantly expressed Lhc proteins. The previously undetected Lhcb7 gene encodes a novel plant Lhcb-type protein that possibly contains an additional, fourth, transmembrane N-terminal helix with a highly conserved motif. As the Lhcb4.3 gene seems to be present only in Eurosid species and as its regulation pattern varies significantly from that of Lhcb4.1 and Lhcb4.2, we conclude it to encode a distinct Lhc protein type, Lhcb8.

  16. Discovering co-occurring patterns and their biological significance in protein families

    PubMed Central

    2014-01-01

    Background The large influx of biological sequences poses the importance of identifying and correlating conserved regions in homologous sequences to acquire valuable biological knowledge. These conserved regions contain statistically significant residue associations as sequence patterns. Thus, patterns from two conserved regions co-occurring frequently on the same sequences are inferred to have joint functionality. A method for finding conserved regions in protein families with frequent co-occurrence patterns is proposed. The biological significance of the discovered clusters of conserved regions with co-occurrences patterns can be validated by their three-dimensional closeness of amino acids and the biological functionality found in those regions as supported by published work. Methods Using existing algorithms, we discovered statistically significant amino acid associations as sequence patterns. We then aligned and clustered them into Aligned Pattern Clusters (APCs) corresponding to conserved regions with amino acid conservation and variation. When one APC frequently co-occured with another APC, the two APCs have high co-occurrence. We then clustered APCs with high co-occurrence into what we refer to as Co-occurrence APC Clusters (Co-occurrence Clusters). Results Our results show that for Co-occurrence Clusters, the three-dimensional distance between their amino acids is closer than average amino acid distances. For the Co-occurrence Clusters of the ubiquitin and the cytochrome c families, we observed biological significance among the residing amino acids of the APCs within the same cluster. In ubiquitin, the residues are responsible for ubiquitination as well as conventional and unconventional ubiquitin-bindings. In cytochrome c, amino acids in the first co-occurrence cluster contribute to binding of other proteins in the electron transport chain, and amino acids in the second co-occurrence cluster contribute to the stability of the axial heme ligand

  17. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids.

    PubMed

    Das, Jayanta Kumar; Das, Provas; Ray, Korak Kumar; Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as 'FPKATD' and 'Y/FTNEKL' without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids' pattern in different proteins.

  18. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids

    PubMed Central

    Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as ‘FPKATD’ and ‘Y/FTNEKL’ without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids’ pattern in different proteins. PMID:27930687

  19. AC magnetohydrodynamic microfluidic switch

    SciTech Connect

    Lemoff, A V; Lee, A P

    2000-03-02

    A microfluidic switch has been demonstrated using an AC Magnetohydrodynamic (MHD) pumping mechanism in which the Lorentz force is used to pump an electrolytic solution. By integrating two AC MHD pumps into different arms of a Y-shaped fluidic circuit, flow can be switched between the two arms. This type of switch can be used to produce complex fluidic routing, which may have multiple applications in {micro}TAS.

  20. Microfluidic Biochip Design

    NASA Technical Reports Server (NTRS)

    Panzarella, Charles

    2004-01-01

    As humans prepare for the exploration of our solar system, there is a growing need for miniaturized medical and environmental diagnostic devices for use on spacecrafts, especially during long-duration space missions where size and power requirements are critical. In recent years, the biochip (or Lab-on-a- Chip) has emerged as a technology that might be able to satisfy this need. In generic terms, a biochip is a miniaturized microfluidic device analogous to the electronic microchip that ushered in the digital age. It consists of tiny microfluidic channels, pumps and valves that transport small amounts of sample fluids to biosensors that can perform a variety of tests on those fluids in near real time. It has the obvious advantages of being small, lightweight, requiring less sample fluids and reagents and being more sensitive and efficient than larger devices currently in use. Some of the desired space-based applications would be to provide smaller, more robust devices for analyzing blood, saliva and urine and for testing water and food supplies for the presence of harmful contaminants and microorganisms. Our group has undertaken the goal of adapting as well as improving upon current biochip technology for use in long-duration microgravity environments. In addition to developing computational models of the microfluidic channels, valves and pumps that form the basis of every biochip, we are also trying to identify potential problems that could arise in reduced gravity and develop solutions to these problems. One such problem is due to the prevalence of bubbly sample fluids in microgravity. A bubble trapped in a microfluidic channel could be detrimental to the operation of a biochip. Therefore, the process of bubble formation in microgravity needs to be studied, and a model of this process has been developed and used to understand how bubbles develop and move through biochip components. It is clear that some type of bubble filter would be necessary in Space, and

  1. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  2. Macromolecular Crystal Growth by Means of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Spearing, Scott; Monaco, Lisa; Molho, Josh; Spaid, Michael; Brasseur, Mike; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    We have performed a feasibility study in which we show that chip-based, microfluidic (LabChip(TM)) technology is suitable for protein crystal growth. This technology allows for accurate and reliable dispensing and mixing of very small volumes while minimizing bubble formation in the crystallization mixture. The amount of (protein) solution remaining after completion of an experiment is minimal, which makes this technique efficient and attractive for use with proteins, which are difficult or expensive to obtain. The nature of LabChip(TM) technology renders it highly amenable to automation. Protein crystals obtained in our initial feasibility studies were of excellent quality as determined by X-ray diffraction. Subsequent to the feasibility study, we designed and produced the first LabChip(TM) device specifically for protein crystallization in batch mode. It can reliably dispense and mix from a range of solution constituents into two independent growth wells. We are currently testing this design to prove its efficacy for protein crystallization optimization experiments. In the near future we will expand our design to incorporate up to 10 growth wells per LabChip(TM) device. Upon completion, additional crystallization techniques such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility for the International Space Station as well as on the ground.

  3. Microfluidic serial dilution circuit.

    PubMed

    Paegel, Brian M; Grover, William H; Skelley, Alison M; Mathies, Richard A; Joyce, Gerald F

    2006-11-01

    In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipeting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

  4. High-pressure microfluidics

    NASA Astrophysics Data System (ADS)

    Hjort, K.

    2015-03-01

    When using appropriate materials and microfabrication techniques, with the small dimensions the mechanical stability of microstructured devices allows for processes at high pressures without loss in safety. The largest area of applications has been demonstrated in green chemistry and bioprocesses, where extraction, synthesis and analyses often excel at high densities and high temperatures. This is accessible through high pressures. Capillary chemistry has been used since long but, just like in low-pressure applications, there are several potential advantages in using microfluidic platforms, e.g., planar isothermal set-ups, large local variations in geometries, dense form factors, small dead volumes and precisely positioned microstructures for control of reactions, catalysis, mixing and separation. Other potential applications are in, e.g., microhydraulics, exploration, gas driven vehicles, and high-pressure science. From a review of the state-of-art and frontiers of high pressure microfluidics, the focus will be on different solutions demonstrated for microfluidic handling at high pressures and challenges that remain.

  5. Development of a fast thermal response microfluidic system using liquid metal

    NASA Astrophysics Data System (ADS)

    Gao, Meng; Gui, Lin

    2016-07-01

    Room temperature liquid metal gallium alloy has been widely used in many micro-electromechanical systems applications, such as on-chip electrical microheaters, micro temperature sensors, micro pumps and so on. Injecting liquid metal into microchannels can provide a simple, rapid, low-cost but efficient way to integrate these elements in microfluidic chips with high accuracy. The liquid metal-filled microstructures can be designed in any shape and easily integrated into microfluidic chips. In this paper, an on-chip liquid metal-based thermal microfluidic system is proposed for quick temperature control at the microscale. The micro system utilizes just one microfluidic chip as a basic working platform, which has liquid metal-based on-chip heaters, temperature sensors and electroosmotic flow pumps. Under the comprehensive control of these elements, the micro system can quickly change the temperature of a target fluid in the microfluidic chip. These liquid metal-based on-chip elements are very helpful for the fabrication and miniaturization of the microfluidic chip. In this paper, deionized water is used to test the temperature control performance of the thermal microfluidic system. According to the experimental results, the micro system can efficiently control the temperature of water ranging from 28 °C to 90 °C. The thermal microfluidic system has great potential for use in many microfluidic applications, such as on-chip polymerase chain reaction, temperature gradient focusing, protein crystallization and chemical synthesis.

  6. Accumulation of BSA in Packed-bed Microfluidics

    NASA Astrophysics Data System (ADS)

    Summers, Samantha; Hu, Chuntian; Hartman, Ryan

    2012-11-01

    Alzheimers and Parkinsons are two diseases that are associated with protein deposition in the brain, causing loss of either cognitive or muscle functioning. Protein deposition diseases are considered progressive diseases since the continual aggregation of protein causes the patient's symptoms to slowly worsen over time. There are currently no known means of treatment for protein deposition diseases. Our goal is to understand the potential for packed-bed microfluidics to study protein accumulation. Measurement of the resistance to flow through micro-scale packed-beds is critical to understanding the process of protein accumulation. Aggregation in bulk is fundamentally different from accumulation on surfaces. Our study attempts to distinguish between either mechanism. The results from our experiments involving protein injection through a microfluidic system will be presented and discussed. Funding received by NSF REU Grant 1062611.

  7. Ethanol and Cancer Induce Similar Changes on Protein Expression Pattern of Human Fibroblast Cell

    PubMed Central

    Zamanian–Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Rezaei Tavirani, Majid

    2016-01-01

    Ethanol has a vast consumption around the world. Many researches confirmed some adverse effect of this component on human health. In addition, recent studies showed significant alteration in both cellular population, and protein profile of human foreskin fibroblast cell line (HFFF2) in the specific dosage of ethanol. Here, the role and interaction of some proteins (characterized by significant alteration in expression due to ethanol effect) analyzed by proteomics and evaluated by considering cancerous case. 2D-electrophoresis findings of comparison of normal fibroblast cells and treated fibroblast with 270 mM dosage of ethanol analyzed by using SameSpots software, R software, and Cytoscape for protein-protein interaction (PPI) investigation. Six proteins with significantly altered expression associated with fundamental properties in a cell identified in ethanol-treated sample. These include AnnexinA5, Heterogeneous nuclear ribonucleoprotein A1, Rho-GDP dissociation inhibitor, Cathepsin L, Cu/Zn-SOD, Rho-GDP dissociation inhibitor, and Serpin peptidase inhibitor. Surprisingly, all these proteins were down-regulated and this pattern is similar to nasopharyngeal carcinoma-associated stromal fibroblast sample. Additionally, protein-protein interaction (PPI) indicates that HNRNPA1, SERPINE1 are hub proteins. Once their expression alters, it can impose vast changes on other molecular function. Based on this approach, ethanol may target same pathways that are related to cancer onset. In addition, some epidemiologic studies proved that ethanol consumption is related to increment of cancer risk. Therefore, more investigation is required in this regard to elicit the feasible relationship. PMID:28228815

  8. From particle self-assembly to functionalized sub-micron protein patterns

    NASA Astrophysics Data System (ADS)

    Blättler, T. M.; Binkert, A.; Zimmermann, M.; Textor, M.; Vörös, J.; Reimhult, E.

    2008-02-01

    Biologically relevant nanopatterns are useful platforms to address fundamental questions, for example, regarding protein-protein and cell-protein interactions. For the creation of nanopatterns, complex and expensive instrumentation is often needed. We present a simple but versatile patterning method using a combination of particle and subsequent molecular self-assembly to produce ordered structures in the micron and sub-micron range. Polystyrene particles were, in a first step, assembled via dip-coating or dried in a drying cell. Silicon wafers and glass slides coated with SiO2 and a top layer of 11 nm of TiO2 were used as substrates. Large hexagonally ordered particle monolayers were formed with high reproducibility. These were subsequently shrunk in a controlled manner by exposure to a O2/N2 plasma and subsequently used as etching masks to transfer the particle pattern onto the substrate, creating TiO2 features in an SiO2 background. After removing the mask the oxide contrast was translated in three simple dip-and-rinse steps into a biochemical contrast of protein-coated features in an inert background. In short, alkane phosphates were first selectively adsorbed to the TiO2 features. Then the SiO2 background was backfilled using poly(L-lysine)-graft-poly(ethylene glycol) and finally streptavidin was adsorbed to the hydrophobic alkane phosphate SAMs, allowing subsequent binding and hybridization of biotinylated DNA.

  9. Plasma protein adsorption patterns on surfaces of Amphotericin B-containing fat emulsions.

    PubMed

    Schmidt, Sven; Müller, Rainer H

    2003-03-18

    Nephrotoxicity of the conventional Amphotericin B formulation Fungizone is the most common side effect in treatment of systemic fungal infections. Lipid formulations of Amphotericin B including fat emulsions showed a reduced nephrotoxicity. In vivo distribution studies of lipid formulations have shown an accumulation of Amphotericin B in liver and spleen, while concentration in the kidneys is reduced. Blood proteins adsorbed onto particles after intravenous administration are regarded as the key factors determining their in vivo fate. Two-dimensional polyacrylamid gel electrophoresis is a powerful tool for analysis of protein adsorption patterns. This paper deals with the question if there is any correlation between proteins adsorbed on surfaces of AmB fat emulsions produced with a new production technique and the potentially organ distribution of this formulation.

  10. Regulation of gene expression in prediapausing embryos of the silkworm, Bombyx mori: pattern of protein synthesis.

    PubMed

    Dorel, C; Coulon, M

    1988-03-01

    Specific qualitative and quantitative changes in protein synthesis occur from the fertilization to the onset of diapause in the silkworm. We have used two-dimensional gel electrophoresis to analyse the patterns of proteins synthesized in prediapausing eggs of Bombyx. This analysis has been carried out with in vivo labelled polypeptides and with proteins synthesized in vitro by RNA isolated at different stages. The oocyte contains an abundant supply of diverse mRNA which are translatable in vitro. A set of proteins with molecular weight range of 68,000 to 74,000 and isoelectric points of 5.85-5.95 (hereafter referred to as No. 30) is specific of the germ-anlage stage. Transcripts encoding the No. 30 proteins are not detectable in oocytes, and inhibition of transcription by actinomycin D indicates that No. 30 mRNA are synthesized de novo. Treating eggs at the germ-anlage stage with 4 N HCl at 46 degrees C prevents diapause and is accompanied by overproduction of No. 30 protein. The induction of No. 30 synthesis is also the main event of the heat shock response. The implications of these findings in relation to early embryonic development and prevention of diapause are discussed.

  11. Mixing in microfluidic devices and enhancement methods

    PubMed Central

    Ward, Kevin; Fan, Z Hugh

    2015-01-01

    Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channel’s hydraulic diameter, flow velocity, and solution’s kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types. PMID:26549938

  12. Mixing in microfluidic devices and enhancement methods.

    PubMed

    Ward, Kevin; Fan, Z Hugh

    2015-09-01

    Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channel's hydraulic diameter, flow velocity, and solution's kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types.

  13. Mixing in microfluidic devices and enhancement methods

    NASA Astrophysics Data System (ADS)

    Ward, Kevin; Fan, Z. Hugh

    2015-09-01

    Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channel’s hydraulic diameter, flow velocity, and solution’s kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types.

  14. Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

    PubMed

    Dellarole, Mariano; Caro, Jose A; Roche, Julien; Fossat, Martin; Barthe, Philippe; García-Moreno E, Bertrand; Royer, Catherine A; Roumestand, Christian

    2015-07-29

    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

  15. Electronic Sensing for Microfluidic Devices

    DTIC Science & Technology

    2005-10-08

    D. J. Insect cell culture in microfluidic channels. Biomedical Microdevices 4, 161-166 (2002). 8 20. Walker, G. M., Zeringue, H. C. & Beebe, D. J...engineering. Biomedical Microdevices 4, 167-175 (2002). 23. Moorthy, J. & Beebe, D. J. Organic and biomimetic designs for microfluidic systems

  16. Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns.

    PubMed

    Park, Yungki; Helms, Volkhard

    2006-09-01

    The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.

  17. Co-integrated microfluidic and THz functions for biochip devices

    NASA Astrophysics Data System (ADS)

    Laurette, S.; Treizebre, A.; Bocquet, B.

    2011-06-01

    TeraHertz (THz) spectroscopy is becoming an alternative way to probe biological interactions in real-time conditions. However, accurate and reproducible THz measurements of aqueous solutions, largely represented in life sciences, remain difficult. A THz microsystem which couples both electromagnetic and microfluidic integrated functions is presented here. Its technological process is accurately detailed and enables easy designs of advanced THz and microfluidic functions. It is composed of the deposition of gold wires on a glass wafer to guide the THz waves. Then, a whole silicon wafer is bonded by using a thermosensitive-polymer thermo-compression. Silicon is deep-etched to create the microchannels which are finally covered with a second glass wafer. This bonding-etching process enables huge freedom and independence for electromagnetic and microfluidic designs. The technological process characterization has shown that the manufactured biochip is compatible with pressures up to 37 bar. First measurements with empty and water-filled channels have been carried out and have shown the ability to perform THz spectroscopy inside the chip. Then, first measurements on proteins have been performed and shown the system ability to probe protein concentration. This kind of microfluidic microsystem, allowing complex design for integrated electronic and microfluidic circuits, defines a true new instrumental way for life science investigations.

  18. Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase*

    PubMed Central

    Fonseca-Maldonado, Raquel; Vieira, Davi Serradella; Alponti, Juliana Sanchez; Bonneil, Eric; Thibault, Pierre; Ward, Richard John

    2013-01-01

    Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex8–16GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions. PMID:23846692

  19. Expression Pattern and Subcellular Localization of the Ovate Protein Family in Rice

    PubMed Central

    Yu, Hui; Jiang, Wenzhu; Liu, Qing; Zhang, Hui; Piao, Mingxin; Chen, Zhengdao; Bian, Mingdi

    2015-01-01

    The Arabidopsis ovate family proteins (AtOFPs) have been shown to function as transcriptional repressors and regulate multiple aspects of plant growth and development. There are 31 genes that encode the full-length OVATE-domain containing proteins in the rice genome. In this study, the gene structure analysis revealed that OsOFPs are intron poor. Phylogenetic analysis suggested that OVATE proteins from rice, Arabidopsis and tomato can be divided into 4 groups (I–IV). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis identified OsOFPs with different tissue-specific expression patterns at all stages of development in the rice plant. Interestingly, nearly half of the total number of OsOFP family was more highly expressed during the seed developmental stage. In addition, seed developmental cis-elements were found in the promoter region of the OsOFPs. Subcellular localization analysis revealed that YFP-OsOFP fusion proteins predominantly localized in the nucleus. Our results suggest that OsOFPs may act as regulatory proteins and play pivotal roles in the growth and development of rice. PMID:25760462

  20. Reconfigurable microfluidic pump enabled by opto-electrical-thermal transduction

    NASA Astrophysics Data System (ADS)

    Takeuchi, Masaru; Hagiwara, Masaya; Haulot, Gauvain; Ho, Chih-Ming

    2013-10-01

    Flexible integration of a microfluidic system comprising pumps, valves, and microchannels was realized by an optoelectronic reconfigurable microchannels (OERM) technique. Projecting a low light fluidic device pattern—e.g., pumps, valves, and channels—onto an OERM platform generates Joule heating and melts the substrate in the bright area on the platform; thus, the fluidic system can be reconfigured by changing the projected light pattern. Hexadecane was used as the substrate of the microfluidic system. The volume change of hexadecane during the liquid-solid phase transition was utilized to generate pumping pressure. The system can pump nanoliters of water within several seconds.

  1. The microfluidic puzzle: chip-oriented rapid prototyping.

    PubMed

    Lim, Jiseok; Maes, Florine; Taly, Valérie; Baret, Jean-Christophe

    2014-05-21

    We demonstrate a new concept for reconfigurable microfluidic devices from elementary functional units. Our approach suppresses the need for patterning, soft molding and bonding when details on a chip have to be modified. Our system has two parts, a base-platform used as a scaffold and functional modules which are combined by 'plug-and-play'. To demonstrate that our system sustains typical pressures in microfluidic experiments, we produce droplets of different sizes using T-junction modules with three different designs assembled successively on a 3 × 3 modular scaffold.

  2. Microfluidic vascular channels in gels using commercial 3D printers

    NASA Astrophysics Data System (ADS)

    Selvaganapathy, P. Ravi; Attalla, Rana

    2016-03-01

    This paper details the development of a three dimensional (3D) printing system with a modified microfluidic printhead used for the generation of complex vascular tissue scaffolds. The print-head features an integrated coaxial nozzle that allows the fabrication of hollow, calcium-polymerized alginate tubes that can easily be patterned using 3Dbioprinting techniques. This microfluidic design allows the incorporation of a wide range of scaffold materials as well as biological constituents such as cells, growth factors, and ECM material. With this setup, gel constructs with embedded arrays of hollow channels can be created and used as a potential substitute for blood vessel networks.

  3. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    PubMed Central

    Wu, Fabai; van Schie, Bas G.C.; Keymer, Juan E.; Dekker, Cees

    2016-01-01

    The boundary of a cell defines the shape and scale for its subcellular organisation. However, the effects of the cell’s spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shape Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2x1x1 to 11x6x1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe, and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale in adaptation to the cell size within a characteristic length range of 3–6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling, and an adaptive range, when and only when facilitated by the three-dimensional confinement of cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood. PMID:26098227

  4. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    NASA Astrophysics Data System (ADS)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  5. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra.

    PubMed

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-10-15

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein-protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors.

  6. Exploration of microfluidic devices based on multi-filament threads and textiles: A review

    PubMed Central

    Nilghaz, A.; Ballerini, D. R.; Shen, W.

    2013-01-01

    In this paper, we review the recent progress in the development of low-cost microfluidic devices based on multifilament threads and textiles for semi-quantitative diagnostic and environmental assays. Hydrophilic multifilament threads are capable of transporting aqueous and non-aqueous fluids via capillary action and possess desirable properties for building fluid transport pathways in microfluidic devices. Thread can be sewn onto various support materials to form fluid transport channels without the need for the patterned hydrophobic barriers essential for paper-based microfluidic devices. Thread can also be used to manufacture fabrics which can be patterned to achieve suitable hydrophilic-hydrophobic contrast, creating hydrophilic channels which allow the control of fluids flow. Furthermore, well established textile patterning methods and combination of hydrophilic and hydrophobic threads can be applied to fabricate low-cost microfluidic devices that meet the low-cost and low-volume requirements. In this paper, we review the current limitations and shortcomings of multifilament thread and textile-based microfluidics, and the research efforts to date on the development of fluid flow control concepts and fabrication methods. We also present a summary of different methods for modelling the fluid capillary flow in microfluidic thread and textile-based systems. Finally, we summarized the published works of thread surface treatment methods and the potential of combining multifilament thread with other materials to construct devices with greater functionality. We believe these will be important research focuses of thread- and textile-based microfluidics in future. PMID:24086179

  7. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

  8. Microfluidic paper-based biomolecule preconcentrator based on ion concentration polarization.

    PubMed

    Han, Sung Il; Hwang, Kyo Seon; Kwak, Rhokyun; Lee, Jeong Hoon

    2016-06-21

    Microfluidic paper-based analytical devices (μPADs) for molecular detection have great potential in the field of point-of-care diagnostics. Currently, a critical problem being faced by μPADs is improving their detection sensitivity. Various preconcentration processes have been developed, but they still have complicated structures and fabrication processes to integrate into μPADs. To address this issue, we have developed a novel paper-based preconcentrator utilizing ion concentration polarization (ICP) with minimal addition on lateral-flow paper. The cation selective membrane (i.e., Nafion) is patterned on adhesive tape, and this tape is then attached to paper-based channels. When an electric field is applied across the Nafion, ICP is initiated to preconcentrate the biomolecules in the paper channel. Departing from previous paper-based preconcentrators, we maintain steady lateral fluid flow with the separated Nafion layer; as a result, fluorescent dyes and proteins (FITC-albumin and bovine serum albumin) are continuously delivered to the preconcentration zone, achieving high preconcentration performance up to 1000-fold. In addition, we demonstrate that the Nafion-patterned tape can be integrated with various geometries (multiplexed preconcentrator) and platforms (string and polymer microfluidic channel). This work would facilitate integration of various ICP devices, including preconcentrators, pH/concentration modulators, and micro mixers, with steady lateral flows in paper-based platforms.

  9. Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease

    PubMed Central

    Torres, Mauricio; Cartier, Luis; Matamala, José Manuel; Hernández, Nancy; Woehlbier, Ute; Hetz, Claudio

    2012-01-01

    Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression

  10. A light writable microfluidic "flash memory": optically addressed actuator array with latched operation for microfluidic applications.

    PubMed

    Hua, Zhishan; Pal, Rohit; Srivannavit, Onnop; Burns, Mark A; Gulari, Erdogan

    2008-03-01

    This paper presents a novel optically addressed microactuator array (microfluidic "flash memory") with latched operation. Analogous to the address-data bus mediated memory address protocol in electronics, the microactuator array consists of individual phase-change based actuators addressed by localized heating through focused light patterns (address bus), which can be provided by a modified projector or high power laser pointer. A common pressure manifold (data bus) for the entire array is used to generate large deflections of the phase change actuators in the molten phase. The use of phase change material as the working media enables latched operation of the actuator array. After the initial light "writing" during which the phase is temporarily changed to molten, the actuated status is self-maintained by the solid phase of the actuator without power and pressure inputs. The microfluidic flash memory can be re-configured by a new light illumination pattern and common pressure signal. The proposed approach can achieve actuation of arbitrary units in a large-scale array without the need for complex external equipment such as solenoid valves and electrical modules, which leads to significantly simplified system implementation and compact system size. The proposed work therefore provides a flexible, energy-efficient, and low cost multiplexing solution for microfluidic applications based on physical displacements. As an example, the use of the latched microactuator array as "normally closed" or "normally open" microvalves is demonstrated. The phase-change wax is fully encapsulated and thus immune from contamination issues in fluidic environments.

  11. Direct Write Protein Patterns for Multiplexed Cytokine Detection From Live Cells Using Electron Beam Lithography

    PubMed Central

    Lau, Uland Y.; Saxer, Sina S.; Lee, Juneyoung; Bat, Erhan; Maynard, Heather D.

    2016-01-01

    Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nano- scale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates allowing for micro- and nano-scale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and anti-tumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications. PMID:26679368

  12. Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw.

    PubMed

    Lee, Jiae; Peng, Yun; Lin, Wen-Yang; Parrish, Jay Z

    2015-01-01

    The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, we screened for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, we identified mutations that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning.

  13. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    PubMed

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  14. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  15. Motion in microfluidic ratchets.

    PubMed

    Caballero, D; Katuri, J; Samitier, J; Sánchez, S

    2016-11-15

    The ubiquitous random motion of mesoscopic active particles, such as cells, can be "rectified" or directed by embedding the particles in systems containing local and periodic asymmetric cues. Incorporated on lab-on-a-chip devices, these microratchet-like structures can be used to self-propel fluids, transport particles, and direct cell motion in the absence of external power sources. In this Focus article we discuss recent advances in the use of ratchet-like geometries in microfluidics which could open new avenues in biomedicine for applications in diagnosis, cancer biology, and bioengineering.

  16. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  17. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin

    PubMed Central

    Riddle, Nicole C.; Minoda, Aki; Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Gorchakov, Andrey A.; Jaffe, Jacob D.; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E.; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Park, Peter J.; Elgin, Sarah C.R.; Karpen, Gary H.

    2011-01-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the “silencing” marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin–heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both “activation” marks (e.g., H3K4me3 and H3K36me3) and “silencing” marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin. PMID:21177972

  18. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

    PubMed

    Riddle, Nicole C; Minoda, Aki; Kharchenko, Peter V; Alekseyenko, Artyom A; Schwartz, Yuri B; Tolstorukov, Michael Y; Gorchakov, Andrey A; Jaffe, Jacob D; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I; Pirrotta, Vincenzo; Park, Peter J; Elgin, Sarah C R; Karpen, Gary H

    2011-02-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

  19. Gray-scale photolithography using microfluidic photomasks

    PubMed Central

    Chen, Chihchen; Hirdes, Danny; Folch, Albert

    2003-01-01

    The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a “photomask” (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and/or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches. PMID:12574512

  20. Differentially photo-crosslinked polymers enable self-assembling microfluidics

    PubMed Central

    Jamal, Mustapha; Zarafshar, Aasiyeh M.; Gracias, David H.

    2012-01-01

    An important feature of naturally self-assembled systems such as leaves and tissues is that they are curved and have embedded fluidic channels that enable the transport of nutrients to, or removal of waste from, specific three-dimensional (3D) regions. Here, we report the self-assembly of photopatterned polymers, and consequently microfluidic devices, into curved geometries. We discovered that differentially photo-crosslinked SU-8 films spontaneously and reversibly curved upon film de-solvation and re-solvation. Photolithographic patterning of the SU-8 films enabled the self-assembly of cylinders, cubes, and bidirectionally folded sheets. We integrated polydimethylsiloxane (PDMS) microfluidic channels with these SU-8 films to self-assemble curved microfluidic networks. PMID:22068594

  1. Fabrication of microfluidic systems in poly(dimethylsiloxane).

    PubMed

    McDonald, J C; Duffy, D C; Anderson, J R; Chiu, D T; Wu, H; Schueller, O J; Whitesides, G M

    2000-01-01

    Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

  2. Integrated microfluidic linking chip for scanning probe nanolithography

    NASA Astrophysics Data System (ADS)

    Ryu, Kee Suk; Wang, Xuefeng; Shaikh, Kashan; Bullen, David; Goluch, Edgar; Zou, Jun; Liu, Chang; Mirkin, Chad A.

    2004-07-01

    This letter reports an architecture for a microfluidic chip that dresses (inks) multiple nanolithography tips in a high-density array in a parallel and multiplexed fashion. The microfluidic chip consists of multiple precision patterned thin-film poly(dimethylsiloxane) (PDMS) patches serving as porous inking pads. Inking chemicals are supplied from loading reservoirs to the inking pads through microfluidic channels. The gas-permeable thin PDMS membranes allow ink molecules to diffuse through while preventing bulk liquid from overflowing or evaporating. The inking chip provides high-density inking, easy loading of inks, and reduced evaporation losses. We present the fabrication process and inking of scanning probe contact printing probes and commercial nitride probes.

  3. Fabrication of polyimide based microfluidic channels for biosensor devices

    NASA Astrophysics Data System (ADS)

    Zulfiqar, Azeem; Pfreundt, Andrea; Svendsen, Winnie Edith; Dimaki, Maria

    2015-03-01

    The ever-increasing complexity of the fabrication process of Point-of-care (POC) devices, due to high demand of functional versatility, compact size and ease-of-use, emphasizes the need of multifunctional materials that can be used to simplify this process. Polymers, currently in use for the fabrication of the often needed microfluidic channels, have limitations in terms of their physicochemical properties. Therefore, the use of a multipurpose biocompatible material with better resistance to the chemical, thermal and electrical environment, along with capability of forming closed channel microfluidics is inevitable. This paper demonstrates a novel technique of fabricating microfluidic devices using polyimide (PI) which fulfills the aforementioned properties criteria. A fabrication process to pattern microfluidic channels, using partially cured PI, has been developed by using a dry etching method. The etching parameters are optimized and compared to those used for fully cured PI. Moreover, the formation of closed microfluidic channel on wafer level by bonding two partially cured PI layers or a partially cured PI to glass with high bond strength has been demonstrated. The reproducibility in uniformity of PI is also compared to the most commonly used SU8 polymer, which is a near UV sensitive epoxy resin. The potential applications of PI processing are POC and biosensor devices integrated with microelectronics.

  4. Microfluidic integrated acoustic waving for manipulation of cells and molecules.

    PubMed

    Barani, Alireza; Paktinat, Hossein; Janmaleki, Mohsen; Mohammadi, Aminollah; Mosaddegh, Peiman; Fadaei-Tehrani, Alireza; Sanati-Nezhad, Amir

    2016-11-15

    Acoustophoresis with its simple and low-cost fabrication, rapid and localized fluid actuation, compatibility with microfluidic components, and biocompatibility for cellular studies, has been extensively integrated into microfluidics to provide on-chip microdevices for a variety of applications in biology, bioengineering and chemistry. Among different applications, noninvasive manipulation of cells and biomolecules are significantly important, which are addressed by acoustic-based microfluidics. Here in this paper, we briefly explain the principles and different configurations of acoustic wave and acoustic streaming for the manipulation of cells and molecules and overview its applications for single cell isolation, cell focusing and sorting, cell washing and patterning, cell-cell fusion and communication, and tissue engineering. We further discuss the application of acoustic-based microfluidic systems for the mixing and transport of liquids, manipulation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, followed by explanation on the present challenges of acoustic-based microfluidics for the handling of cells and molecules, and highlighting the future directions.

  5. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  6. Fabrication of a Paper-Based Microfluidic Device to Readily Determine Nitrite Ion Concentration by Simple Colorimetric Assay

    ERIC Educational Resources Information Center

    Wang, Bo; Lin, Zhiqiang; Wang, Min

    2015-01-01

    Paper-based microfluidic devices (µPAD) are a burgeoning platform of microfluidic analysis technology. The method described herein is for use in undergraduate and high school chemistry laboratories. A simple and convenient µPAD was fabricated by easy patterning of filter paper using a permanent marker pen. The usefulness of the device was…

  7. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori.

    PubMed

    Gong, Da-Ping; Zhang, Hui-Jie; Zhao, Ping; Lin, Ying; Xia, Qing-You; Xiang, Zhong-Huai

    2007-03-01

    Insect chemosensory proteins (CSPs) as well as odorant-binding proteins (OBPs) have been supposed to transport hydrophobic chemicals to receptors on sensory neurons. Compared with OBPs, CSPs are expressed more broadly in various insect tissues. We performed a genome-wide analysis of the candidate CSP gene family in the silkworm. A total of 20 candidate CSPs, including 3 gene fragments and 2 pseudogenes, were characterized based on their conserved cysteine residues and their similarity to CSPs in other insects. Some of these genes were clustered in the silkworm genome. The gene expression pattern of these candidates was investigated using RT-PCR and microarray, and the results showed that these genes were expressed primarily in mature larvae and the adult moth, suggesting silkworm CSPs may be involved in development. The majority of silkworm CSP genes are expressed broadly in tissues including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes.

  8. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

    PubMed

    Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

    2010-02-05

    The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

  9. Nanostructured microfluidic digestion system for rapid high-performance proteolysis.

    PubMed

    Cheng, Gong; Hao, Si-Jie; Yu, Xu; Zheng, Si-Yang

    2015-02-07

    A novel microfluidic protein digestion system with a nanostructured and bioactive inner surface was constructed by an easy biomimetic self-assembly strategy for rapid and effective proteolysis in 2 minutes, which is faster than the conventional overnight digestion methods. It is expected that this work would contribute to rapid online digestion in future high-throughput proteomics.

  10. Three-dimensional surface microfluidics enabled by spatiotemporal control of elastic fluidic interface.

    PubMed

    Hong, Lingfei; Pan, Tingrui

    2010-12-07

    As an emerging alternative to the conventional counterpart, surface microfluidics incorporates both intrinsic resistive solid-liquid and elastic frictionless gas-liquid interfaces, leading to unique flow-pressure characteristics. Furthermore, the open-surface microfluidic platforms can be fabricated on a monolithic substrate with high wettability contrast by the previously reported one-step lithographic process of a photosensitive superhydrophobic nanocomposite material, which permits flexible fluidic operations and direct surface modifications. In the paper, we first present three-dimensional microfluidic manipulations utilizing the unconventional gas-liquid interfaces of surface microfluidics, outlined by the micropatterned wetting boundaries (also known as the triple lines). In contrast to the primary linear (resistive) nature of the conventional closed-channel microfluidics, the distinct elastic interface of surface microfluidics enables remarkable three-dimensional (deformable) and time-dependent (capacitive) operations of the flow. Specifically, spatiotemporal dependence of microflow patterns on the planar fluidic surfaces has been theoretically analyzed and experimentally characterized. Utilizing the unconventional interface-enabled flow-pressure relationship, novel surface fluidic operations, including microflow regulation and flow-controlled switching, have been demonstrated and fully investigated. Furthermore, three-dimensional surface microfluidic networks together with analog-to-digital stereo-flow activations have been established, in which miniature capillary bridges form fluidic connections between two independent surface microfluidic circuits.

  11. Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts

    SciTech Connect

    Garling, D.J.; Tassava, R.A.

    1984-08-01

    Polyacrylamide slab gel electrophoresis and (/sup 35/S)methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.

  12. Fabricating PFPE Membranes for Microfluidic Valves and Pumps

    NASA Technical Reports Server (NTRS)

    Greer, Frank; White, Victor E.; Lee, Michael C.; Willis, Peter A.; Grunthaner, Frank J.; Rolland, Jason; Rolland, Jason

    2009-01-01

    A process has been developed for fabricating membranes of a perfluoropolyether (PFPE) and integrating them into valves and pumps in laboratory-on-achip microfluidic devices. Membranes of poly(tetrafluoroethylene) [PTFE] and poly(dimethylsilane) [PDMS] have been considered for this purpose and found wanting. By making it possible to use PFPE instead of PTFE or PDMS, the present process expands the array of options for further development of microfluidic devices for diverse applications that could include detection of biochemicals of interest, detection of toxins and biowarfare agents, synthesis and analysis of proteins, medical diagnosis, and synthesis of fuels.

  13. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  14. Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

    PubMed

    González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; González-Flores, Oscar; Galván-Rosas, Agustín; Porfirio Gómora-Arrati; Camacho-Arroyo, Ignacio

    2014-01-01

    Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

  15. Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

    SciTech Connect

    Bloom, T. )

    1991-03-01

    Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development. In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with ({sup 32}P)orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.

  16. Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode.

    PubMed

    Bax, Daniel V; Tipa, Roxana S; Kondyurin, Alexey; Higgins, Michael J; Tsoutas, Kostadinos; Gelmi, Amy; Wallace, Gordon G; McKenzie, David R; Weiss, Anthony S; Bilek, Marcela M M

    2012-07-01

    The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation.

  17. Fabrication of Self-Cleaning, Reusable Titania Templates for Nanometer and Micrometer Scale Protein Patterning.

    PubMed

    Moxey, Mark; Johnson, Alexander; El-Zubir, Osama; Cartron, Michael; Dinachali, Saman Safari; Hunter, C Neil; Saifullah, Mohammad S M; Chong, Karen S L; Leggett, Graham J

    2015-06-23

    The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of reuseable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). With the use of a Lloyd's mirror two-beam interferometer, self-assembled monolayers of alkylphosphonates adsorbed on the native oxide of a Ti film were patterned by photocatalytic nanolithography. In regions exposed to a maximum in the interferogram, the monolayer was removed by photocatalytic oxidation. In regions exposed to an intensity minimum, the monolayer remained intact. After exposure, the sample was etched in piranha solution to yield Ti nanostructures with widths as small as 30 nm. NIL was performed by using a silicon stamp to imprint a spin-cast film of titanium dioxide resin; after calcination and reactive ion etching, TiO2 nanopillars were formed. For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times. These simple methods appear to yield durable, reuseable samples that may be of value to laboratories that require nanostructured biological interfaces but do not have access to the infrastructure required for nanofabrication.

  18. Microfluidic Biochip Design

    NASA Technical Reports Server (NTRS)

    Panzarella, Charles

    2004-01-01

    As humans prepare for the exploration of our solar system, there is a growing need for miniaturized medical and environmental diagnostic devices for use on spacecrafts, especially during long-duration space missions where size and power requirements are critical. In recent years, the biochip (or Lab-on-a-Chip) has emerged as a technology that might be able to satisfy this need. In generic terms, a biochip is a miniaturized microfluidic device analogous to the electronic microchip that ushered in the digital age. It consists of tiny microfluidic channels, pumps and valves that transport small amounts of sample fluids to biosensors that can perform a variety of tests on those fluids in near real time. It has the obvious advantages of being small, lightweight, requiring less sample fluids and reagents and being more sensitive and efficient than larger devices currently in use. Some of the desired space-based applications would be to provide smaller, more robust devices for analyzing blood, saliva and urine and for testing water and food supplies for the presence of harmful contaminants and microorganisms. Our group has undertaken the goal of adapting as well as improving upon current biochip technology for use in long-duration microgravity environments.

  19. Jamming in Microfluidic Channels

    NASA Astrophysics Data System (ADS)

    Ortiz, Carlos; Daniels, Karen; Riehn, Robert

    2009-11-01

    We experimentally investigate the flow of a colloid through a microfluidic device. The glass microfluidic device consists of a wide channel with spatially periodic funnels manufactured with photolithographic methods. The device was etched to a depth of about 1 micron that restricts the solid phase of the colloid, fluorescent polystyrene spheres with sub-micron radii, to quasi-2D motion. The liquid phase of the colloid is an aqueous solution with trace amounts of a non-ionic surfactant and with a pH about 2 units above the pKa of the surface groups on the polystyrene spheres to maintain a stable colloid at concentrations high enough to produce jamming. The flow rate of the colloid is controlled by a computer interfaced syringe pump with two controllable modes of operation: a continuous, steady mode that provides a plug-like velocity profile and a discrete, jerky mode that sends compressional waves of specifiable sizes through the colloid. Using fluorescence microscopy, we observe the interactions between the colloid and the glass funnels and investigate how the interaction depends on the funnel geometry. In particular, we investigate the jamming transition from a liquid-like flowing state to a solid-like stationary state.

  20. Active micromachines: Microfluidics powered by mesoscale turbulence

    PubMed Central

    Thampi, Sumesh P.; Doostmohammadi, Amin; Shendruk, Tyler N.; Golestanian, Ramin; Yeomans, Julia M.

    2016-01-01

    Dense active matter, from bacterial suspensions and microtubule bundles driven by motor proteins to cellular monolayers and synthetic Janus particles, is characterized by mesoscale turbulence, which is the emergence of chaotic flow structures. By immersing an ordered array of symmetric rotors in an active fluid, we introduce a microfluidic system that exploits spontaneous symmetry breaking in mesoscale turbulence to generate work. The lattice of rotors self-organizes into a spin state where neighboring discs continuously rotate in permanent alternating directions due to combined hydrodynamic and elastic effects. Our virtual prototype demonstrates a new research direction for the design of micromachines powered by the nematohydrodynamic properties of active turbulence. PMID:27419229

  1. Precise and automated microfluidic sample preparation.

    SciTech Connect

    Crocker, Robert W.; Patel, Kamlesh D.; Mosier, Bruce P.; Harnett, Cindy K.

    2004-07-01

    Autonomous bio-chemical agent detectors require sample preparation involving multiplex fluid control. We have developed a portable microfluidic pump array for metering sub-microliter volumes at flowrates of 1-100 {micro}L/min. Each pump is composed of an electrokinetic (EK) pump and high-voltage power supply with 15-Hz feedback from flow sensors. The combination of high pump fluid impedance and active control results in precise fluid metering with nanoliter accuracy. Automated sample preparation will be demonstrated by labeling proteins with fluorescamine and subsequent injection to a capillary gel electrophoresis (CGE) chip.

  2. Microfluidic Exosome Analysis toward Liquid Biopsy for Cancer.

    PubMed

    He, Mei; Zeng, Yong

    2016-08-01

    Assessment of a tumor's molecular makeup using biofluid samples, known as liquid biopsy, is a prominent research topic in precision medicine for cancer, due to its noninvasive property allowing repeat sampling for monitoring molecular changes of tumors over time. Circulating exosomes recently have been recognized as promising tumor surrogates because they deliver enriched biomarkers, such as proteins, RNAs, and DNA. However, purification and characterization of these exosomes are technically challenging. Microfluidic lab-on-a-chip technology effectively addresses these challenges owing to its inherent advantages in integration and automation of multiple functional modules, enhancing sensing performance, and expediting analysis processes. In this article, we review the state-of-the-art development of microfluidic technologies for exosome isolation and molecular characterization with emphasis on their applications toward liquid biopsy-based analysis of cancer. Finally, we share our perspectives on current challenges and future directions of microfluidic exosome analysis.

  3. Improving agglutination tests by working in microfluidic channels.

    PubMed

    Degré, G; Brunet, E; Dodge, A; Tabeling, P

    2005-06-01

    Latex agglutination tests are used for the diagnosis of diseases in man and animals. They are generally simple, cheap, and do not require sophisticated equipment, nor highly specialized skills. In this Technical Note, we put latex agglutination tests in a microfluidic format. The experiment is performed in PDMS (polydimethylsiloxane) microchannels, using streptavidin-coated superparamagnetic beads and a magnetic field. The target molecule is biotinylated protein A. By taking full advantage of the microfluidic conditions (scaling down of the detection volume and controlled action of the shear flow), we achieved an analytical sensitivity of 10 fmol l(-1)(several hundreds of fg ml(-1)) and a fast response (a few minutes) ; the test is also quantitative. Performances of agglutination tests can thus be improved by orders of magnitude by adapting them to a microfluidic format; this comes in addition to the usual advantages offered by this technology (integration, high throughput etc.).

  4. Microfluidic chip-based technologies: emerging platforms for cancer diagnosis

    PubMed Central

    2013-01-01

    The development of early and personalized diagnostic protocols is considered the most promising avenue to decrease mortality from cancer and improve outcome. The emerging microfluidic-based analyzing platforms hold high promises to fulfill high-throughput and high-precision screening with reduced equipment cost and low analysis time, as compared to traditional bulky counterparts in bench-top laboratories. This article overviewed the potential applications of microfluidic technologies for detection and monitoring of cancer through nucleic acid and protein biomarker analysis. The implications of the technologies in cancer cytology that can provide functional personalized diagnosis were highlighted. Finally, the future niches for using microfluidic-based systems in tumor screening were briefly discussed. PMID:24070124

  5. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra

    PubMed Central

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-01-01

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein–protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors. PMID:26468920

  6. Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.

    PubMed

    Kecskemeti, Adam; Bako, Jozsef; Csarnovics, Istvan; Csosz, Eva; Gaspar, Attila

    2017-03-15

    Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

  7. Correlations Between Single Cell Signaling Dynamics and Protein Expressions Profiles

    DTIC Science & Technology

    2005-08-16

    the need for fluorescent or radioactive labels. These deter- minations have been performed within picoliter volumes using microfluidic channels...developments are addressing this. Future efforts will fully integrate the microfluidic nanophysiometer, OCIBD analyte detection system, MALDI-TOF protein...upon full integration of the microfluidic nanophysiometer, OCIBD analyte detection system, MALDI-TOF protein traps, and cell loading (for internalization

  8. Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

    PubMed Central

    Xue, Niannan; Li, Xia; Bertulli, Cristina; Li, Zhaoying; Patharagulpong, Atipat; Sadok, Amine; Huang, Yan Yan Shery

    2014-01-01

    Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among

  9. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor.

  10. A Robust and Engineerable Self-Assembling Protein Template for the Synthesis and Patterning of Ordered Nanoparticle Arrays

    NASA Technical Reports Server (NTRS)

    McMillan, R. Andrew; Howard, Jeanie; Zaluzec, Nestor J.; Kagawa, Hiromi K.; Li, Yi-Fen; Paavola, Chad D.; Trent, Jonathan D.

    2004-01-01

    Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here we introduce a general technique for patterning materials on the nanoscale using genetically modified protein cage structures called chaperonins that self-assemble into crystalline templates. Constrained chemical synthesis of transition metal nanoparticles is specific to templates genetically functionalized with poly-Histidine sequences. These arrays of materials are ordered by the nanoscale structure of the crystallized protein. This system may be easily adapted to pattern a variety of materials given the rapidly growing list of peptide sequences selected by screening for specificity for inorganic materials.

  11. Microfluidic technology for molecular diagnostics.

    PubMed

    Robinson, Tom; Dittrich, Petra S

    2013-01-01

    Molecular diagnostics have helped to improve the lives of millions of patients worldwide by allowing clinicians to diagnose patients earlier as well as providing better ongoing therapies. Point-of-care (POC) testing can bring these laboratory-based techniques to the patient in a home setting or to remote settings in the developing world. However, despite substantial progress in the field, there still remain many challenges. Progress in molecular diagnostics has benefitted greatly from microfluidic technology. This chapter aims to summarise the more recent advances in microfluidic-based molecular diagnostics. Sections include an introduction to microfluidic technology, the challenges of molecular diagnostics, how microfluidic advances are working to solve these issues, some alternative design approaches, and detection within these systems.

  12. Emergence of microfluidic wearable technologies.

    PubMed

    Yeo, Joo Chuan; Kenry; Lim, Chwee Teck

    2016-10-18

    There has been an intense interest in the development of wearable technologies, arising from increasing demands in the areas of fitness and healthcare. While still at an early stage, incorporating microfluidics in wearable technologies has enormous potential, especially in healthcare applications. For example, current microfluidic fabrication techniques can be innovatively modified to fabricate microstructures and incorporate electrically conductive elements on soft, flexible and stretchable materials. In fact, by leverarging on such microfabrication and liquid manipulation techniques, the developed flexible microfluidic wearable technologies have enabled several biosensing applications, including in situ sweat metabolites analysis, vital signs monitoring, and gait analysis. As such, we anticipate further significant breakthroughs and potential uses of wearable microfluidics in active drug delivery patches, soft robotics sensing and control, and even implantable artificial organs in the near future.

  13. Many Local Pattern Texture Features: Which Is Better for Image-Based Multilabel Human Protein Subcellular Localization Classification?

    PubMed Central

    Xu, Ying-Ying; Shen, Hong-Bin

    2014-01-01

    Human protein subcellular location prediction can provide critical knowledge for understanding a protein's function. Since significant progress has been made on digital microscopy, automated image-based protein subcellular location classification is urgently needed. In this paper, we aim to investigate more representative image features that can be effectively used for dealing with the multilabel subcellular image samples. We prepared a large multilabel immunohistochemistry (IHC) image benchmark from the Human Protein Atlas database and tested the performance of different local texture features, including completed local binary pattern, local tetra pattern, and the standard local binary pattern feature. According to our experimental results from binary relevance multilabel machine learning models, the completed local binary pattern, and local tetra pattern are more discriminative for describing IHC images when compared to the traditional local binary pattern descriptor. The combination of these two novel local pattern features and the conventional global texture features is also studied. The enhanced performance of final binary relevance classification model trained on the combined feature space demonstrates that different features are complementary to each other and thus capable of improving the accuracy of classification. PMID:25050396

  14. Towards printable open air microfluidics.

    SciTech Connect

    Collord, Andrew; Cook, Adam W.; Clem, Paul Gilbert; Fenton, Kyle Ross; Apblett, Christopher Alan; Branson, Eric D.

    2010-04-01

    We have demonstrated a novel microfluidic technique for aqueous media, which uses super-hydrophobic materials to create microfluidic channels that are open to the atmosphere. We have demonstrated the ability to perform traditional electrokinetic operations such as ionic separations and electrophoresis using these devices. The rate of evaporation was studied and found to increase with decreasing channel size, which places a limitation on the minimum size of channel that could be used for such a device.

  15. Passive microfluidic array card and reader

    DOEpatents

    Dugan, Lawrence Christopher; Coleman, Matthew A.

    2011-08-09

    A microfluidic array card and reader system for analyzing a sample. The microfluidic array card includes a sample loading section for loading the sample onto the microfluidic array card, a multiplicity of array windows, and a transport section or sections for transporting the sample from the sample loading section to the array windows. The microfluidic array card reader includes a housing, a receiving section for receiving the microfluidic array card, a viewing section, and a light source that directs light to the array window of the microfluidic array card and to the viewing section.

  16. Pen microfluidics: rapid desktop manufacturing of sealed thermoplastic microchannels

    PubMed Central

    Rahmanian, Omid

    2013-01-01

    A unique technique for the rapid fabrication of thermoplastic microfluidic chips is described. The method enables the realization of fully-sealed microchannels in around one hour while requiring only minimal infrastructure by taking advantage of a solvent swelling mechanism that allows raised features to be patterned on the surface of homogeneous thermoplastic materials. Patterning is achieved without photolithography by simply drawing the desired microchannel pattern onto the polymer surface using a suitable ink as a masking layer, either manually or under robotic control, followed by timed exposure to solvent vapor to yield a desired depth for the masked channel features. The channels are then permanently sealed through solvent bonding of the microchannel chip to a mating thermoplastic substrate. The process is demonstrated using cyclic olefin copolymer as a thermoplastic material, with fully operational microfluidic devices fabricated following a true desktop manufacturing model suitable for rapid prototyping. PMID:23344819

  17. A glass microfluidic chip adhesive bonding method at room temperature

    NASA Astrophysics Data System (ADS)

    Pan, Yu-Jen; Yang, Ruey-Jen

    2006-12-01

    This paper presents a novel method using UV epoxy resin for the bonding of glass blanks and patterned plates at room temperature. There is no need to use a high-temperature thermal fusion process and therefore avoid damaging temperature-sensitive metals in a microchip. The proposed technique has the further advantage that the sealed glass blanks and patterned plates can be separated by the application of adequate heat. In this way, the microchip can be opened, the fouling microchannels may be easily cleaned-up and the plates then re-bonded to recycle the microchip. The proposed sealing method is used to bond a microfluidic device, and the bonding strength is then investigated in a series of chemical resistance tests conducted in various chemicals. Leakage of solution was evaluated in a microfluidic chip using pressure testing to 1.792 × 102 kPa (26 psi), and the microchannel had no observable leak. Electrical leakage between channels was tested by comparing the resistances of two bonding methods, and the result shows no significant electrical leakage. The performance of the device obtained from the proposed bonding method is compared with that of the thermal fusion bonding technique for an identical microfluidic device. It is found that identical results are obtained under the same operating conditions. The proposed method provides a simple, quick and inexpensive method for sealing glass microfluidic chips.

  18. Two different immunostaining patterns of beta-amyloid precursor protein (APP) may distinguish traumatic from nontraumatic axonal injury.

    PubMed

    Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

    2015-09-01

    Immunostaining for beta-amyloid precursor protein (APP) is recognized as an effective tool for detecting traumatic axonal injury, but it also detects axonal injury due to ischemic or other metabolic causes. Previously, we reported two different patterns of APP staining: labeled axons oriented along with white matter bundles (pattern 1) and labeled axons scattered irregularly (pattern 2) (Hayashi et al. (Leg Med (Tokyo) 11:S171-173, 2009). In this study, we investigated whether these two patterns are consistent with patterns of trauma and hypoxic brain damage, respectively. Sections of the corpus callosum from 44 cases of blunt head injury and equivalent control tissue were immunostained for APP. APP was detected in injured axons such as axonal bulbs and varicose axons in 24 of the 44 cases of head injuries that also survived for three or more hours after injury. In 21 of the 24 APP-positive cases, pattern 1 alone was observed in 14 cases, pattern 2 alone was not observed in any cases, and both patterns 1 and 2 were detected in 7 cases. APP-labeled injured axons were detected in 3 of the 44 control cases, all of which were pattern 2. These results suggest that pattern 1 indicates traumatic axonal injury, while pattern 2 results from hypoxic insult. These patterns may be useful to differentiate between traumatic and nontraumatic axonal injuries.

  19. Microfluidic serial dilution ladder.

    PubMed

    Ahrar, Siavash; Hwang, Michelle; Duncan, Philip N; Hui, Elliot E

    2014-01-07

    Serial dilution is a fundamental procedure that is common to a large number of laboratory protocols. Automation of serial dilution is thus a valuable component for lab-on-a-chip systems. While a handful of different microfluidic strategies for serial dilution have been reported, approaches based on continuous flow mixing inherently consume larger amounts of sample volume and chip real estate. We employ valve-driven circulatory mixing to address these issues and also introduce a novel device structure to store each stage of the dilution process. The dilution strategy is based on sequentially mixing the rungs of a ladder structure. We demonstrate a 7-stage series of 1 : 1 dilutions with R(2) equal to 0.995 in an active device area of 1 cm(2).

  20. Enhanced Microfluidic Electromagnetic Measurements

    NASA Technical Reports Server (NTRS)

    Giovangrandi, Laurent (Inventor); Ricco, Antonio J. (Inventor); Kovacs, Gregory (Inventor)

    2015-01-01

    Techniques for enhanced microfluidic impedance spectroscopy include causing a core fluid to flow into a channel between two sheath flows of one or more sheath fluids different from the core fluid. Flow in the channel is laminar. A dielectric constant of a fluid constituting either sheath flow is much less than a dielectric constant of the core fluid. Electrical impedance is measured in the channel between at least a first pair of electrodes. In some embodiments, enhanced optical measurements include causing a core fluid to flow into a channel between two sheath flows of one or more sheath fluids different from the core fluid. An optical index of refraction of a fluid constituting either sheath flow is much less than an optical index of refraction of the core fluid. An optical property is measured in the channel.

  1. Inertial Focusing in Microfluidics

    PubMed Central

    Martel, Joseph M.; Toner, Mehmet

    2015-01-01

    When Segré and Silberberg in 1961 witnessed particles in a laminar pipe flow congregating at an annulus in the pipe, scientists were perplexed and spent decades learning why such behavior occurred, finally understanding that it was caused by previously unknown forces on particles in an inertial flow. The advent of microfluidics opened a new realm of possibilities for inertial focusing in the processing of biological fluids and cellular suspensions and created a field that is now rapidly expanding. Over the past five years, inertial focusing has enabled high-throughput, simple, and precise manipulation of bodily fluids for a myriad of applications in point-of-care and clinical diagnostics. This review describes the theoretical developments that have made the field of inertial focusing what it is today and presents the key applications that will make inertial focusing a mainstream technology in the future. PMID:24905880

  2. Inertial focusing in microfluidics.

    PubMed

    Martel, Joseph M; Toner, Mehmet

    2014-07-11

    When Segré and Silberberg in 1961 witnessed particles in a laminar pipe flow congregating at an annulus in the pipe, scientists were perplexed and spent decades learning why such behavior occurred, finally understanding that it was caused by previously unknown forces on particles in an inertial flow. The advent of microfluidics opened a new realm of possibilities for inertial focusing in the processing of biological fluids and cellular suspensions and created a field that is now rapidly expanding. Over the past five years, inertial focusing has enabled high-throughput, simple, and precise manipulation of bodily fluids for a myriad of applications in point-of-care and clinical diagnostics. This review describes the theoretical developments that have made the field of inertial focusing what it is today and presents the key applications that will make inertial focusing a mainstream technology in the future.

  3. Microfluidic large scale integration of viral-host interaction analysis.

    PubMed

    Ben-Ari, Ya'ara; Glick, Yair; Kipper, Sarit; Schwartz, Nika; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron

    2013-06-21

    Viral-host interactions represent potential drug targets for novel antiviral strategies (Flisiak et al., Hepatology, 2008, 47, 817-26). Hence, it is important to establish an adequate platform for identifying and analyzing such interactions. In this review, we discuss bottlenecks in conventional protein-protein interaction methodologies and present the contribution of innovative microfluidic-based technologies towards a solution to these problems with respect to viral-host proteomics.

  4. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292